CTL peptide epitopes and antigen-specific t cells, methods for their discovery, and uses thereof

ABSTRACT

The present invention relates to CTL peptide epitopes, high-throughput methods for their identification, and their uses. In particular, the present invention relates to peptide epitopes for cancer immunotherapy and Hepatitis C Virus vaccines. The present invention also relates to methods and systems for identifying antigen-specific CTLs.

FIELD OF THE INVENTION

The present invention relates to Cytotoxic T cell (also known ascytotoxic T lymphocytes, CTL) peptide epitopes, high-throughput methodsfor their identification, and their uses. In particular, the presentinvention relates to peptide epitopes for cancer immunotherapy andHepatitis C Virus (HCV) vaccines. The present invention also relates tomethods and systems for identifying antigen-specific CTLs.

BACKGROUND OF THE INVENTION

Cytotoxic T cells (CTLs), selectively kill target cells that expressdefined peptides in complex with major histocompatibility complex (MHC)class I molecules on the cell surface. Most tumor-associated antigens(TAA) are wild type self-proteins, and T cells that recognize peptidesfrom these antigens with high affinity are deleted during thymicdevelopment. Thus, the utility of T cells for detection of self-peptidespresented on self-HLA is limited by tolerance. The number of epitopesidentified from TAA after two decades of intense research amounts toless than 600 (1). The ability to rapidly identify new CTL epitopesfacilitates the development of effective immunotherapeutic strategiesagainst cancer.

MHC molecules can be isolated from cells, and the associated peptideseluted for identification by mass spectrometry (MS). Ultimately, thisapproach may provide a description of the entire MHC-bound peptiderepertoire; the immunopeptidome (2, 3). This is, however, a dauntingtask, and it is unclear whether current MS-based protocols provide therequired sensitivity. Indeed, although 100,000-750,000 peptide-MHC classI complexes are expressed for each allelic product on the cell surface(for HLA-A and HLA-B loci) (3, 4), the largest HLA-ligandome identifiedto date contains 14,065 peptides (5). In contrast, the predicted numberof different HLA class I ligands would be 352.000 using thewell-renowned computer algorithm NetMHCpan, considering that on average4.4% of all nonamers bind HLA class I (6) and that a cell contains atleast 8×10⁶ distinct nonamers (7). Thus, there is a very large gapbetween the number of confirmed and predicted HLA ligands. In addition,it is not known which fraction of the confirmed HLA ligands are actuallyfunctional epitopes and can stimulate a CTL response.

It is therefore crucial to identify additional potent CTL epitopes, inorder to develop new and effective immunotherapy strategies.Consequently, methods enabling efficient identification of functionalepitopes are urgently needed.

SUMMARY OF THE INVENTION

The present invention relates to CTL peptide epitopes, high-throughputmethods for their identification, and their uses. In particular, thepresent invention relates to peptide epitopes for cancer immunotherapyand Hepatitis C Virus (HCV) vaccines. The present invention also relatesto methods and systems for identifying CTL peptide epitopes.

In some embodiments, the present invention provides a method ofidentifying cytotoxic T cell epitopes from an antigen of interest,comprising: a) expressing at least one candidate antigen and a definedHLA molecule (e.g., HLA-A*02:01), in an antigen presenting cell, orexpressing at least one candidate antigen in an antigen-presenting cellthat is naturally positive for the defined HLA; b) utilizing anaffinity-based algorithmic prediction to identify peptides from thecandidate antigen that are predicted to bind to the defined HLA molecule(e.g., HLA-A*02:01); c) complexing each of the predicted peptides withthe HLA molecule (e.g., HLA-A*02:01) to generate synthetic peptide:HLAcomplexes; d) contacting the antigen-presenting cells with T cells thatare not tolerant to the peptide-HLA complex to induce T cell responsesagainst the complexes; and e) contacting the synthetic peptide:HLAmultimers with the T cells to directly identify epitopes that react withthe antigen presenting cells.

In some embodiments, the present invention provides a method ofidentifying cytotoxic T cell epitopes from an antigen of interest,comprising: a) expressing at least one candidate antigen and a definedHLA molecule (e.g., HLA-A*02:01), in an antigen presenting cell, orexpressing at least one candidate antigen in an antigen-presenting cellthat is naturally positive for the defined HLA; b) utilizing anaffinity-based algorithmic prediction to identify peptides from thecandidate antigen that are predicted to bind to the defined HLA molecule(e.g., HLA-A*02:01); c) contacting the antigen-presenting cells with Tcells that are not tolerant to the peptide-HLA complex to induce T cellresponses against the complexes; d) complexing each of the predictedpeptides with the HLA molecule (e.g., HLA-A*02:01) to generate syntheticpeptide:HLA complexes; and e) contacting the synthetic peptide:HLAmultimers with the T cells to directly identify T cells reactive to thepredicted peptide-HLA complexes.

In some embodiments, cells that express the defined HLA molecule and thepredicted peptides are used to stimulate the induced T cells to directlyidentify responding T cells among induced T cells by means of forinstance cytokine production, upregulation of membrane markers ordegranulation.

In some embodiments, the complexes are multimerized to increase bindingstrength (e.g., to generate HLAp multimers). In some embodiments wherethe peptide antigen is foreign to the T cells (e.g. HCV-derivedpeptides) the defined HLA molecule is further expressed by the T cells(e.g., self-HLA molecule). In some embodiments where the peptide antigenis derived from a self-protein (e.g., CD20 or myeloperoxidase), thedefined HLA molecule is not expressed by the T cells (e.g., foreignHLA-molecule). Thus, in both situations, the peptide-HLA complex isrecognized as foreign by the T cells as either the peptide or the HLAmolecule is foreign to the T cells. In some embodiments, the candidatetarget proteins are screened for cell-type specific expression in normaland disease-affected cells prior to use in the epitope identificationassay. In some embodiments, the complexing of peptides withHLA-molecules for subsequent generation of peptide:HLA multimerscomprises UV-induced ligand exchange and multimerization. In someembodiments, the complexes are labeled.

In further embodiments, the present invention provides methods ofidentifying cytotoxic T cell epitopes from an antigen of interest,comprising: a) expressing at least one candidate self-antigen in anantigen presenting cell that expresses a defined HLA molecule; b)utilizing an affinity-based algorithmic prediction to identify aplurality of peptides from the candidate antigen that are predicted tobind to the defined HLA molecule to provide predicted peptides; c)synthesizing the predicted peptides and forming complexes of thesynthesized predicted peptides with the defined HLA molecule to generatepredicted peptide:defined HLA molecule complexes; d) contacting theantigen-presenting cells with T cells that lack expression of thedefined HLA antigen and therefore are not tolerant to the predictedpeptide:defined HLA molecule complexes to provide induced T cells; ande) identifying T cells reactive with the predicted peptide:defined HLAmolecule complexes by contacting the induced T cells with the predictedpeptide:defined HLA molecule complexes to identify T cells reactive withthe complexes.

In still further embodiments, the present invention provides methods ofidentifying cytotoxic T cell epitopes from an antigen of interest,comprising: a) expressing at least one candidate foreign antigen in anantigen presenting cell that expresses a defined HLA molecule; b)utilizing an affinity-based algorithmic prediction to identify aplurality of peptides from the candidate antigen that are predicted tobind to the defined HLA molecule to provide predicted peptides; c)complexing the predicted peptides with the defined HLA molecule togenerate peptide:defined HLA molecule complexes; d) contacting theantigen-presenting cells with T cells that are not tolerant to thepeptide:defined HLA molecule complexes to induce T cell responsesagainst the complexes in vitro; and e) identifying T cells reactive withthe predicted peptide:defined HLA molecule complexes by contacting theinduced T cells with the predicted peptide:defined HLA moleculecomplexes to identify T cells reactive with the complexes.

In some embodiments, the defined HLA molecule is naturally expressed inthe antigen presenting cell. In some embodiments, the defined HLAmolecule is exogenously expressed in the antigen presenting cell. Insome embodiments, the defined HLA molecule is HLA-A*02:01. In someembodiments, the candidate foreign antigen is expressed by transfectionof mRNA encoding the antigens. In some embodiments, the candidateforeign antigen is foreign to the T cells and the defined HLA moleculeis expressed by the T cells. In some embodiments, the candidate foreignantigen is an HCV antigen. In some embodiments, the candidate foreignantigen is derived from a self-protein and the defined HLA molecule isnot expressed by the T cells. In some embodiments, the candidate foreignantigen is CD20 or myeloperoxidase. In some embodiments, the candidateforeign antigen is screened for cell-type specific expression in normaland disease-affected cells prior to the expressing.

In some embodiments, the complexing comprises UV-induced ligand exchangeand multimerization. In some embodiments, the complexes are labeled. Insome embodiments, the identification comprises identifying reactive Tcells by assaying cytokine production following stimulation of theinduced T cells with the antigen presenting cells. In some embodiments,the identification comprises identifying reactive T cells by assayingupregulation of membrane markers following stimulation of the induced Tcells with the antigen presenting cells. In some embodiments, theidentification comprises identifying reactive T cells by assayingdegranulation following stimulation of the induced T cells with theantigen presenting cells. In some embodiments, the methods furthercomprise the step of cloning a T-cell receptor from one of theidentified T-cells and modifying T-cells isolated from a patient toexpress the T-cell receptor. In some embodiments, the methods furthercomprise administering the modified T cells to a patient who naturallyexpresses the defined HLA molecule.

It will be understood that the candidate foreign antigens and HLAmolecules may be expressed in the antigen presenting cells by a varietyof methods known in the art, including, but not limited to, transfectionwith RNA, transfection with DNA expression constructs, transfection withplasmid expression constructs, and transduction with viral expressionconstructs. In some embodiments the viral expression construct is anadenoassociated or a lentiviral vector. Electroporation and other meansknown in the art for introducing nucleic acids into cells may also beused for expressing the candidate foreign antigen and HLA molecules.

In some embodiments, the methods of the present invention preferablyutilize predicted peptides with a specified predicted binding affinity,for example, a predicted KD of less than 500 nm, 300 nm, 200 nm, 100 nm,or preferable less than 50 nm or 20 nm or 10 nm.

Additional embodiments provide CTL epitopes identified by aforementionedmethods, for use in identification of therapeutically relevant T cellsand T-cell receptors.

In some embodiments, the present invention provides a method of treatingcancer, comprising, isolating peripheral blood cells; and activating theT-cells (e.g., CTL), to recognize one or more epitopes (e.g., those inTables 1 or 2 (SEQ ID Nos:1-50)) from cell-type specific self-proteinsin the context of a defined foreign HLA molecule; expanding them toincrease the cell numbers; and transfusing them back to the patient.

In some embodiments, the present invention provides a method of a)generating T cells by the aforementioned method; b) isolating theT-cells; c) performing T cell receptor cloning and sequenceidentification: and d) genetically modifying T cells isolated from apatient ex vivo to express the T cell receptor(s); and e) transfusingthe modified T-cells back to the patient.

In some embodiments, T-cells recognizing one of the epitopes in Tables 1or 2 (SEQ ID Nos:1-50) are expanded by contacting them with a syntheticpeptide of Tables 1 or 2 (SEQ ID Nos:1-50) in cell culture whencomplexed with the defined HLA molecule expressed on antigen-presentingcells. In some embodiments, one or more cytokine (e.g., IL-2) isadministered with the activated T-cells.

In some embodiments, the present invention provides methods of treatingcancer in a subject who naturally expresses a defined HLA molecule,comprising: a) isolating peripheral blood comprising T-cells from adonor lacking the HLA molecule, b) activating the T-cells to recognizeone or more epitopes identified by the methods described above bycontacting the T-cells with a peptide antigen complexed with the HLAmolecule expressed on antigen-presenting cells; c) expanding the T-cellsto provide an expanded population of T cells followed by isolation of Tcells in the expanded population of T-cells that recognize thepeptide-HLA complex; and d) administering the isolated T-cells to thepatient. In some embodiments, the T-cells are CTL. In some embodiments,the peptide antigen is selected from the group consisting of SEQ IDNos:1-50 and a peptide from a cell-type specific self protein. In someembodiments, one or more cytokines are administered concurrently withthe activated T-cell. In some embodiments, the cytokine in IL-2. In someembodiments, a peptide selected from the group consisting of SEQ IDNOs:1-50 is administered with the activated T-cells. In someembodiments, the isolation comprises contacting the T cells with solublemultimers of the peptide antigen complexed with the HLA molecule.

In some embodiments, the T cells or T cell receptors of the presentinvention are used in combination with other treatment modalities,including but not limited to small molecule drugs (chemotherapy agents),antibodies and vaccines. The combination therapy may be eitherconcurrent or sequential.

In some embodiments, the present invention provides a method of treatinginfection by a microorganism (e.g. HCV infection), comprising: a)isolating peripheral blood cells; b) activating T-cells in the bloodcells (e.g., CTL) to recognize one or more disease specific epitopes(e.g., those in Table 3 (SEQ ID Nos:51-70)) using the aforementionedmethod; c) expanding T-cells recognizing the epitope to increase thecell numbers; and d) transfusing them back to the patient. In someembodiments, the T cells recognizing the epitope are isolated for T cellreceptor cloning and sequence identification, and one or more of the Tcell receptors are used to genetically modify patient T cells outsidethe patient to express the T cell receptor(s), and subsequentlytransfused back to the patient.

In some embodiments, the present invention provides methods of treatinginfection by a microorganism in a subject naturally expressing a definedHLA molecule, comprising: a) isolating peripheral blood comprisingT-cells from the subject, b) activating the T-cells to recognize one ormore epitopes by contacting the T-cells with a peptide antigen complexedwith the HLA molecule expressed by antigen-presenting cells; c)expanding the T-cells to increase the number of cells; and d)administering the activated T-cells to the patient. In some embodiments,the T-cells are CTL. In some embodiments, the peptide antigen isselected from the group consisting of SEQ ID NOs:51-70. In someembodiments, the microorganism is HCV. In some embodiments, one or morecytokines are administered concurrently with the activated T-cell. Insome embodiments, the cytokine is IL-2. In some embodiments, a peptideselected from the group consisting of SEQ ID NOs:51-70 is administeredwith the activated T-cells.

Embodiments of the present invention provide a vaccine compositioncomprising a peptide comprising one or more immunogens selected from thegroup consisting of the peptides described in Table 3. In someembodiments, the immunogen is covalently bound to a carrier protein(e.g., cholera toxin, pseudomonas exotoxin A, toxoids, virus likeparticles, tetanus toxin/toxoid, diphtheria toxin/toxoid or hepatitis Bsurface protein). In some embodiments, the carrier protein is a sterilepharmaceutically acceptable carrier protein. In some embodiments, thevaccine composition further comprises an adjuvant.

Additional embodiments provide a method of inducing an immune response,comprising administering a vaccine composition described herein to asubject under conditions such that the subject generates an immuneresponse (e.g., T-cell mediated immune response) to a viral target,including but not limited to the HCV protein NS3 and/or Core antigens.

In some embodiments, the immune response treats HCV infection in thesubject.

Further embodiments of the present invention provide a kit comprisingthe vaccine composition described herein. In some embodiments, the kitfurther comprises a device for administration of the vaccine. In someembodiments, the kit further comprises one or more additional components(e.g., including but not limited to, sanitation components, temperaturecontrol components, adjuvants, a physiologically tolerable buffer, orinstructions for using the vaccine composition).

Additional embodiments of the invention are described herein.

DESCRIPTION OF THE FIGURES

FIG. 1. Allo-restricted CTLs recognize 37/50 algorithm-predictedepitopes from the differentiation antigens CD20 and MPO. (A-C) T cellsfrom HLA-A2neg donors were stimulated with HLA-A2-transfected APCs(moDCs for priming and EBV-LCLs for re-stimulation). The APCs wereco-transfected with mRNA transcripts encoding full-length CD20 or MPO,respectively. (A) T cells were primed with APCs transfected with MPO.The dot plots are gated on viable CD8pos T cells and show staining withcolor-coded pHLA-multimers complexed with three different MPO peptides;numbers in parenthesis indicate peptide numbers as shown in (C). Flowcytometric analysis of cells staining positively for multimersconjugated to PE, APC or PerCP-Cy5.5 is shown as red, blue or greendots, respectively. (B,C) T cells were primed with APCs transfected withCD20 (blue bars) or MPO (red bars). Bars show frequencies of cellsstaining positively with pHLA-A2 multimers with indicated peptides fromCD20 (B) or MPO (C) among viable CD8pos T cells. Control multimers(ctrl) were complexed with two irrelevant peptides (MART-1(ELAGIGILTV)(SEQ ID NO:73)) or CMV (NLVPMVATV) (SEQ ID NO:74)) andmixed. Results shown are representative of 2 (CD20) or 4 (MPO)experiments. Horizontal dashed lines indicate cut-off.

FIG. 2. Allo-restricted T cells reactive with CD20-derived epitopesrespond specifically and strongly to HLA-A2posCD20pos target cells. CTLsreactive with individual CD20-HLA-A2 multimers (designated by the numberof the peptide they recognized, see FIG. 1), were sorted and expandedinto cell lines. The cells were co-cultured for 5 h with indicatedtarget cells at E:T ratios of 1:2. (A) Flow cytometric analysis showingpercentage of CD8posHLA-A2neg cells expressing CD107a/b (indicative ofdegranulation) and producing IFN-γ in cell line #4 (Tc-4) whenco-cultured with indicated target cells. (B) The bars show frequenciesof CD107a/b and/or IFN-γ positive events among CD8pos T cells whenindicated CTL line (CD20 Tc-1, 4, 6, 16) was co-cultured with indicatedtarget cell (x-axis). All target cells were HLA-A2pos, either naturallyor by genetic introduction (+A2), as indicated. They were grouped intothose expressing CD20, either naturally (JVM-2, EBV-LCLs) or by geneticmodification (HeLa+A2+CD20), or into CD20 neg target cells that werepeptide loaded (+pep) or not (−pep). Target cells were excluded fromflow cytometric analysis by staining with anti-HLA-A2. Error barsindicate SD of duplicates. All CTL lines were tested in separateexperiments.

FIG. 3. Allo-restricted T cells reactive with MPO-derived epitopesrespond specifically and strongly to HLA-A2posMPOpos target cells.Assays were performed as described in FIG. 2. The bars show frequenciesof CD107a/b and/or IFN-γ positive events among CD8posHLA-A2neg T cellswhen indicated CTL line (MPO Tc-1, 2, 3, 9, 13, 23, 24) was co-culturedwith indicated target cell (x-axis). All target cells were HLA-A2pos,either naturally or by genetic modification (+A2), as indicated. Theywere grouped into those expressing MPO, either naturally (THP-1,monocytes) or induced (HeLa+A2+MPO), or into MPO negative target cellsthat were peptide loaded (+pep) or not (−pep). SupT1 cells were notavailable when performing the experiments for Tc-3 and -13. Target cellswere excluded from flow cytometric analysis by staining withanti-HLA-A2. Error bars indicate SD of duplicates. All CTL lines weretested in separate experiments.

FIG. 4. Platform for high-throughput epitope discovery. (1) Candidatetarget proteins were screened for cell-type specific expression innormal and malignant hematopoietic cells (GeneSapiens.org). (2) Selectedtarget proteins, and HLA-A*02:01, were cloned into a vector for in vitroproduction of mRNA encoding full-length protein. (3) Monocyte-deriveddendritic cells from HLA-A2neg donors were transfected with the mRNA,and subsequently co-cultured with autologous, non-adherent peripheralblood mononuclear cells. The cultures were re-stimulated with EBV-LCLstransfected with mRNA encoding target protein and HLA-A2 on days 12 and19. (4) Selected targets were subjected to affinity-based algorithmicprediction of HLA-A*02:01-restricted peptides(cbs.dtu.dk/services/NetMHC/) and those predicted to bind with thehighest affinities were synthesized. (5) Peptides were complexed withHLA-A*02:01 monomers by UV-induced ligand exchange, followed bymultimerization using streptavidin (SA) conjugated to PE, APC orPerCP-Cy5.5, respectively. (6) By end of culture, CD8pos T cellsreactive to different epitopes were detected by flow cytometricmeasurements of combinations of fluorescently labeled pHLA-A2 multimers.

FIG. 5. CTL lines recognizing CD20 or MPO-derived peptides respondstrongly and specifically to HLA-A2pos antigen positive target cells.Assays were performed as described in FIG. 2, except that onlydegranulation responses were measured. The bars show frequencies ofCD107a/b positive cells of CD8pos cells when indicated CTL line wasco-cultured with indicated target cells (x-axis). (A) All fourCD20-reactive CTL lines shown in FIG. 2 were tested for reactivityagainst THP-1 cells in the absence or presence (+pep) of loading withrelevant peptide. (B-E) CTLs reactive to MPO peptides #4 (B) and #25 (C)from the same donor as used in FIG. 3, or from an additional donorreactive to peptides #1, #2, #3, #4 or #9 (D,E), were tested fordegranulation in response to the indicated target cells (x-axis). Alltarget cells expressed HLA-A2, either naturally or induced (+A2), asindicated, and were grouped into target cells expressing MPO, eithernaturally (THP-1, monocytes) or induced (HeLa+A2+MPO, SupT1+A2+MPO), andinto antigen negative target cells loaded (+pep) or not (−pep) withrelevant peptide. The results in A are representative of two experimentsperformed.

FIG. 6. T cell clones reactive to MPO peptide #25 show specificresponses of variable magnitudes to HLA-A2posMPOpos target cells, anddepend on multiple but different amino acids for peptide recognition.(A) Assays were performed as described in FIG. 2, except that onlydegranulation responses were measured. The bars show frequencies ofCD107a/b positive cells of CD8posHLA-A2neg cells when indicated T cellclone was co-cultured with indicated target cells (x-axis). (B) Three ofthe clones shown in (A) were examined for binding of PE-conjugatedHLA-A2 multimers containing the #25 peptide with and without an alaninesubstitution in each position, using flow cytometry. The negativecontrol multimer (Ctrl) incorporated a CMV-derived peptide (NLVPMVATV(SEQ ID NO:75)). (C) Multimers incorporating alanine-replaced or WTpeptides were tested for binding to primary T cells transduced with aMART-1 specific TCR, along with a multimer incorporating the cognateMART-1 peptide (ELAGIGILTV (SEQ ID NO:73) as a positive control or theCMV-derived peptide as a negative control.

FIG. 7. CTL lines recognizing CD20 or MPO respond to low concentrationsof peptide. CTL lines reactive to the MPO-derived peptides #1, 9, 23 or24 (left graph), or to the CD20-derived peptides #1, 4 or 6 (rightgraph) were co-incubated with HLA-A2pos SupT1 cells loaded withindicated concentrations of cognate peptide. The assays were performedas described in FIG. 2. The symbols represent frequencies of CD107a/band/or IFN-γ positive events among CD8pos HLA-A2neg T cells. Error barsrepresent SD of duplicates.

FIG. 8. Recognition of HCV antigens is similar when peptides arepresented on autologous or foreign HLA. (A, B) Monocyte-derived DCs(moDCs) from HLA-A2^(pos) donors were transfected with mRNA encoding HCVcore (left) or NS3 antigens (right). The cells were co-cultured withautologous non-adherent PBMCs. On days 12 and 19, T cells wererestimulated with autologous EBV-LCL transfected with the same HCVantigens. On day 26 co-cultures were stained with CD8 and pHLA-A2multimers with indicated peptides from HCV core (C1-9) and HCV NS3(N53-1-11), respectively, conjugated to PE or APC. Flow cytometricanalysis of percentages of viable CD8^(pos) T cells staining positivelyfor indicated multimers is shown in (A) (black dots), or as bars in (B).Grey and black bars indicate cells obtained from HLA-A2^(pos) donorsco-expressing HLA-B7 or HLA-B8, respectively. Control multimers (ctrl;CMV and MART-1), NS3 pool (mix of multimers containing NS3-1, -2 or -6peptides) and core pool (C1-3) were included to test specificity ofstaining, as was staining of cells from the HLA-B7^(pos) donor with theNS3-10/HLA-B8 multimer, and staining of cells from the HLA-B8^(pos)donor with the C8/HLA-B7 multimer, respectively. (C,D) MoDCs from twoHLA-A2^(neg) donors (grey and black bars, respectively) wereco-transfected with HLA-A2 and core (left) or NS3 antigens (right).Autologous T cells were stimulated, and restimulated with transfectedEBV-LCL and stained as described above. Percentages of viable CD8^(pos)T cells staining positively for indicated multimers are shown in (C)(black dots), or as bars in (D). Horizontal dashed lines indicatecut-off.

FIG. 9: MPO-reactive T cell clones recognize their specific target withhigh avidity. Clones (indicated by number) from cell line number 9 wereco-cultured with the HLA-A2 positive MPO negative cell line SupT1 loadedwith indicated concentrations of the cognate MPO peptide or not (nopep). The clones were also tested for recognition of endogenouslypresented peptide by co-culture with the HLA-A2 positive MPO positivemyeloid leukemia cell line THP-1.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

As used herein, the term “peptide” refers to a polymer of two or moreamino acids joined via peptide bonds or modified peptide bonds. As usedherein, the term “dipeptides” refers to a polymer of two amino acidsjoined via a peptide or modified peptide bond.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the terms“modified”, “mutant”, and “variant” refer to a gene or gene product thatdisplays modifications in sequence and or functional properties (i.e.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally-occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

The term “fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion as compared to thenative protein, but where the remaining amino acid sequence is identicalto the corresponding positions in the amino acid sequence deduced from afull-length cDNA sequence. Fragments typically are at least 4 aminoacids long, preferably at least 20 amino acids long, usually at least 50amino acids long or longer, and span the portion of the polypeptiderequired for intermolecular binding of the compositions with its variousligands and/or substrates.

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, antigens are purified byremoval of contaminating proteins. The removal of contaminants resultsin an increase in the percent of antigen (e.g., antigen of the presentinvention) in the sample.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is thenative protein contains only those amino acids found in the protein asit occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four consecutive amino acid residues tothe entire amino acid sequence minus one amino acid.

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabelled antibodies.

The term “antigenic determinant” as used herein refers to that portionof an antigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies that bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

As used herein, the term “microorganism” refers to any species or typeof microorganism, including but not limited to, bacteria, viruses,archaea, fungi, protozoans, mycoplasma, prions, and parasitic organisms.The term microorganism encompasses both those organisms that are in andof themselves pathogenic to another organism (e.g., animals, includinghumans, and plants) and those organisms that produce agents that arepathogenic to another organism, while the organism itself is notdirectly pathogenic or infective to the other organism.

As used herein the term “pathogen,” and grammatical equivalents, refersto an organism (e.g., biological agent), including microorganisms, thatcauses a disease state (e.g., infection, pathologic condition, disease,etc.) in another organism (e.g., animals and plants) by directlyinfecting the other organism, or by producing agents that causes diseasein another organism (e.g., bacteria that produce pathogenic toxins andthe like). “Pathogens” include, but are not limited to, viruses,bacteria, archaea, fungi, protozoans, mycoplasma, prions, and parasiticorganisms.

The terms “bacteria” and “bacterium” refer to all prokaryotic organisms,including those within all of the phyla in the Kingdom Procaryotae. Itis intended that the term encompass all microorganisms considered to bebacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, andRickettsia. All forms of bacteria are included within this definitionincluding cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc.

As used herein the terms “disease” and “pathologic condition” are usedinterchangeably, unless indicated otherwise herein, to describe adeviation from the condition regarded as normal or average for membersof a species or group (e.g., humans), and which is detrimental to anaffected individual under conditions that are not inimical to themajority of individuals of that species or group. Such a deviation canmanifest as a state, signs, and/or symptoms (e.g., diarrhea, nausea,fever, pain, blisters, boils, rash, immune suppression, inflammation,etc.) that are associated with any impairment of the normal state of asubject or of any of its organs or tissues that interrupts or modifiesthe performance of normal functions. A disease or pathological conditionmay be caused by or result from contact with a microorganism (e.g., apathogen or other infective agent (e.g., a bacterium)), may beresponsive to environmental factors (e.g., malnutrition, industrialhazards, and/or climate), may be responsive to an inherent defect of theorganism (e.g., genetic anomalies) or to combinations of these and otherfactors.

As used herein, the term “adjuvant” refers to any substance that canstimulate an immune response (e.g., a mucosal immune response). Someadjuvants can cause activation of a cell of the immune system (e.g., anadjuvant can cause an immune cell to produce and secrete a cytokine).Examples of adjuvants that can cause activation of a cell of the immunesystem include, but are not limited to, saponins purified from the barkof the Q. saponaria tree, such as QS21 (a glycolipid that elutes in the21st peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc.,Worcester, Mass.); poly(di(carboxylatophenoxy)phosphazene (PCPP polymer;Virus Research Institute, USA); derivatives of lipopolysaccharides suchas monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc.,Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyldipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related tolipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongationfactor (a purified Leishmania protein; Corixa Corporation, Seattle,Wash.). Traditional adjuvants are well known in the art and include, forexample, aluminum phosphate or hydroxide salts (“alum”). In someembodiments, compositions of the present invention (e.g., comprisingnanoemulsion inactivated RSV) are administered with one or moreadjuvants (e.g., to skew the immune response towards a Th1 or Th2 typeresponse).

As used herein, the term “carrier protein” references to a molecule thatinteracts (e.g., via covalent attachment) to an antigen or immunogen. Insome embodiments, carrier proteins enhance the presentation of antigensto the immune system. In some embodiments, the resulting complex engagesthe T cell arm of the immune response, resulting in higher levels ofantibodies and cell response. In some embodiments, carrier proteins aresterile and pharmaceutically acceptable.

As used herein, the term “under conditions such that the subjectgenerates an immune response” refers to any qualitative or quantitativeinduction, generation, and/or stimulation of an immune response (e.g.,innate or acquired).

A used herein, the term “immune response” refers to a response by theimmune system of a subject. For example, immune responses include, butare not limited to, a detectable alteration (e.g., increase) in Tollreceptor activation, lymphokine (e.g., cytokine (e.g., Th1 or Th2 typecytokines) or chemokine) expression and/or secretion, macrophageactivation, dendritic cell activation, T cell activation (e.g., CD4+ orCD8+ T cells), NK cell activation, and/or B cell activation (e.g.,antibody generation and/or secretion). Additional examples of immuneresponses include binding of an immunogen (e.g., antigen (e.g.,immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic Tlymphocyte (“CTL”) response, inducing a B cell response (e.g., antibodyproduction), and/or T-helper lymphocyte response, and/or a delayed typehypersensitivity (DTH) response against the antigen from which theimmunogenic polypeptide is derived, expansion (e.g., growth of apopulation of cells) of cells of the immune system (e.g., T cells, Bcells (e.g., of any stage of development (e.g., plasma cells), andincreased processing and presentation of antigen by antigen presentingcells. An immune response may be to immunogens that the subject's immunesystem recognizes as foreign (e.g., non-self antigens frommicroorganisms (e.g., pathogens), or self-antigens recognized asforeign). Thus, it is to be understood that, as used herein, “immuneresponse” refers to any type of immune response, including, but notlimited to, innate immune responses (e.g., activation of Toll receptorsignaling cascade) cell-mediated immune responses (e.g., responsesmediated by T cells (e.g., antigen-specific T cells) and non-specificcells of the immune system) and humoral immune responses (e.g.,responses mediated by B cells (e.g., via generation and secretion ofantibodies into the plasma, lymph, and/or tissue fluids). The term“immune response” is meant to encompass all aspects of the capability ofa subject's immune system to respond to antigens and/or immunogens(e.g., both the initial response to an immunogen (e.g., a pathogen) aswell as acquired (e.g., memory) responses that are a result of anadaptive immune response).

As used herein, the term “immunity” refers to protection from disease(e.g., preventing or attenuating (e.g., suppression) of a sign, symptomor condition of the disease) upon exposure to a microorganism (e.g.,pathogen) capable of causing the disease Immunity can be innate (e.g.,non-adaptive (e.g., non-acquired) immune responses that exist in theabsence of a previous exposure to an antigen) and/or acquired (e.g.,immune responses that are mediated by B and T cells following a previousexposure to antigen (e.g., that exhibit increased specificity andreactivity to the antigen)).

As used herein, the term “immunogen” refers to an agent (e.g., amicroorganism (e.g., bacterium, virus or fungus) and/or portion orcomponent thereof (e.g., a protein antigen)) that is capable ofeliciting an immune response in a subject. In some embodiments,immunogens elicit immunity against the immunogen (e.g., microorganism(e.g., pathogen or a pathogen product)).

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample. Test compoundscomprise both known and potential therapeutic compounds. A test compoundcan be determined to be therapeutic by screening using the screeningmethods of the present invention. A “known therapeutic compound” refersto a therapeutic compound that has been shown (e.g., through animaltrials or prior experience with administration to humans) to beeffective in such treatment or prevention.

The term “sample” as used herein is used in its broadest sense. In onesense it can refer to a tissue sample. In another sense, it is meant toinclude a specimen or culture obtained from any source, as well asbiological. Biological samples may be obtained from animals (includinghumans) and encompass fluids, solids, tissues, and gases. Biologicalsamples include, but are not limited to blood products, such as plasma,serum and the like. These examples are not to be construed as limitingthe sample types applicable to the present invention. A sample suspectedof containing a human chromosome or sequences associated with a humanchromosome may comprise a cell, chromosomes isolated from a cell (e.g.,a spread of metaphase chromosomes), genomic DNA (in solution or bound toa solid support such as for Southern blot analysis), RNA (in solution orbound to a solid support such as for Northern blot analysis), cDNA (insolution or bound to a solid support) and the like. A sample suspectedof containing a protein may comprise a cell, a portion of a tissue, anextract containing one or more proteins and the like.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to CTL peptide epitopes, high-throughputmethods for their identification, and their uses. In particular, thepresent invention relates to peptide epitopes for cancer immunotherapyand Hepatitis C Virus vaccines. The present invention also relates tomethods and systems for identifying CTL peptide epitopes.

Leukemia/lymphoma are treated with a variety of therapeutic modalities,including chemotherapy, monoclonal antibodies and/or T cell-basedimmunotherapy, in the form of allogeneic hematopoietic stem celltransplantation (AHSCT). Although many patients respond to AHSCT, onlyfew patients are eligible for this therapy due to its hightreatment-related morbidity and mortality. Thus, for large fractions ofthese patients there is no cure, and a large unmet need for improvedtherapeutic options.

Fifty-eighty % of patients fail to develop an appropriate immuneresponse to acute infection with Hepatitis C Virus and develop chronicinfection, which might lead to subsequent liver damage and liver cancer.There is currently no effective prophylactic vaccine for Hepatitis CVirus, and large fractions of patients with chronic infection are notcured by available treatment. There is thus a large unmet need fordevelopment of more effective vaccines/immunotherapy strategies againstHepatitis C Virus infection.

Self-TAA are the main targets in immunotherapy of cancer today. However,only 600 CTL epitopes have been discovered thus far in spite of 20 yearsof research, of which only 250 are from non-melanoma antigens. The mainreason is that T cell responses to various epitopes have been studied inpatients, who are tolerized against self-TAA presented on self-HLA. Theapproach described herein provides a multitude of novel targets fromself-TAA discovered with high sensitivity, which is a huge improvementrelative to approaches used today. Thus, in one study of two proteins werapidly identified 37 CTL epitopes.

Although immunogenic epitopes in Hepatitis C Virus have been extensivelystudied, 5 novel epitopes were discovered in the NS3 antigen, and inaddition a large number of the known epitopes were confirmed.

Accordingly, embodiments of the present disclosure provide novel peptideepitopes to CD20 and MPO that provide targets for cancer immunotherapyof these patients and epitopes to NS3 useful in the generation of animmune response to Hepatitis C virus (HCV).

Today, the discovery of CTL epitopes for use in vaccination andimmunotherapy of cancer (infection) is done by studying cells frompatients with cancer (infection). However, all studies depending onpatient cells for epitope discovery are limited by tolerance to self andprevious immunization history of the individual. In contrast, thestrategy presented here took advantage of induction of responses inT-cell repertoires from antigen-inexperienced, non-tolerizedindividuals. This indicates that the use of self-tolerant T-cellrepertoires, rather than poor peptide presentation, is the reason whyfew cancer epitopes have been previously identified.

CTLs recognize a complex between a peptide, called cancer antigenpeptide, and a major histocompatibility complex class I antigen (MHCclass I antigen, also referred to as HLA antigen) through the T cellreceptors (TCRs), and thereby attacking autologous tumor cells.

In some embodiments, the present invention provides a cancer vaccinethat activates CTL. In some embodiments cancer immunotherapy is ex vivoT-cell mediated immunotherapy (See e.g., Overwijk et al., J Exp Med.2003 Aug. 18; 198(4): 569-580 and Rosenberg et al., Nat Med. 2004September; 10(9): 909-915; each of which is herein incorporated byreference in its entirety). In some embodiments, ex vivo T-cellmedicated cancer vaccines or immunotherapy agents are generated byisolation of either allogenic or autologous immune cells, enriching oractivating them outside the body (e.g., by contacting them with apeptide of Table 1) and transfusing them back to the patient. Theinjected immune cells are highly cytotoxic to the cancer cellsexpressing a particular epitope, targeting cancer cells (See e.g., Maheret al, Br J Cancer. 2004 Aug. 31; 91(5): 817-821; herein incorporated byreference in its entirety). In some embodiments, activated T-cells(e.g., CTL) are administered along with one or more cytokine (e.g.,IL-2).

In some embodiments, cancer therapy combines ex vivo T-cell stimulationand a cancer vaccine (e.g., comprising a peptide described in Table 1).

In some embodiments, cell-based immunotherapy is conducted in vivo byadministering a peptide described herein along with an agent thatstimulates CTLs (e.g., cytokines such as Interleukins). The peptidesdescribed herein have a CTL-inducing ability and such induced CTLs canexert the anti-cancer activity through cytotoxic action or production oflymphokines. The peptides described herein can be used as an activeingredient of cancer vaccine for treating or preventing cancer. Thus,the present invention provides cancer vaccine (pharmaceuticalcomposition as cancer vaccine) comprising as an active ingredient apeptide (e.g., those in Table 1). When cancer vaccine of the presentinvention is administered to a patient, the peptide is presented toantigen-presenting cells. Then, CTLs specifically recognizing thepresented HLA-antigen complex proliferate and destroy the cancer cells,whereby the treatment or prevention of cancer becomes possible. Thecancer vaccine of the present invention can be used in the prevention ortreatment of a variety of cancers (e.g., myeloid leukemia or aB-lymphoid malignancy).

Thus, in another embodiment, the present invention provides a method fortreatment or prevention of cancer, which comprises administering aneffective amount of cancer vaccine of the present invention to a patientin need thereof.

The cancer vaccine comprising as an active ingredient a peptide of thepresent invention may contain a single CTL epitope (e.g., thosedescribed in Table 1) or an epitope peptide wherein a peptide is ligatedwith other peptide(s) (CTL epitope, helper epitope, etc.) as an activeingredient. In some embodiments, multiple peptides are utilized.

The cancer vaccine comprising as an active ingredient a peptide of thepresent invention may be administered together with a pharmaceuticallyacceptable carrier, for example, an appropriate adjuvant, or in the formof particles so that the cellular immunity can be establishedeffectively. As an adjuvant, those described in a literature (Clin.Microbiol. Rev., 7:277-289, 1994), and the like are applicable. Concreteexamples include microorganism-derived components, cytokines,plant-derived components, marine organism-derived components, mineralgels such as aluminium hydroxide, surfactants such as lysolecithin andPluronic polyols, polyanions, peptides, oil emulsion (emulsionpreparations) and the like. Liposomal preparations, particulatepreparations in which the ingredient is bound to beads having a diameterof several μm, preparations in which the ingredient is attached tolipids, and the like, are also contemplated.

Administration may be achieved, for example, intradermally,subcutaneously, intramuscularly, or intravenously. Although the dosageof the peptide of the present invention in the formulation may beadjusted as appropriate depending on the disease to be treated, the ageand the body weight of patient, it is usually within the range of 0.0001mg-1000 mg, preferably 0.001 mg-1000 mg, more preferably 0.1 mg-10 mg,which can be preferably administered once in every several days to everyseveral months.

In some embodiments, the HCV peptide epitopes described herein are usedto generate vaccines. In some embodiments, immunocarriers (e.g., carrierproteins) are attached to immunogens. In some embodiments, carrierproteins are covalently linked to an antigen or immunogen. Exemplarycarrier proteins include, but are not limited to, cholera toxin,pseudomonas exotoxin A, toxoids, virus like particles, tetanustoxin/toxoid, diphtheria toxin/toxoid and hepatitis B surface protein.Additional carrier proteins are known to those of skill in the art.

An effective amount of the present vaccine is one in which a sufficientimmunological response to the vaccine is raised to protect a subjectexposed to HCV or generate a cancer specific immune response.Preferably, the subject is protected to an extent in which from one toall of the adverse physiological symptoms or effects of the disease tobe prevented are found to be significantly reduced.

In some preferred embodiments, the presence of one or more co-factors oragents reduces the amount of immunogen required for induction of animmune response (e.g., a protective immune response (e.g., protectiveimmunization)). In some embodiments, the presence of one or moreco-factors or agents can be used to skew the immune response towards acellular (e.g., T cell mediated) or humoral (e.g., antibody mediated)immune response. The present invention is not limited by the type ofco-factor or agent used in a therapeutic agent of the present invention.

Adjuvants are described in general in Vaccine Design—the Subunit andAdjuvant Approach, edited by Powell and Newman, Plenum Press, New York,1995. The present invention is not limited by the type of adjuvantutilized (e.g., for use in a composition (e.g., pharmaceuticalcomposition). For example, in some embodiments, suitable adjuvantsinclude an aluminium salt such as aluminium hydroxide gel (alum) oraluminium phosphate. In some embodiments, an adjuvant may be a salt ofcalcium, iron or zinc, or may be an insoluble suspension of acylatedtyrosine, or acylated sugars, cationically or anionically derivatizedpolysaccharides, or polyphosphazenes.

In general, an immune response is generated to an antigen through theinteraction of the antigen with the cells of the immune system Immuneresponses may be broadly categorized into two categories: humoral andcell mediated immune responses (e.g., traditionally characterized byantibody and cellular effector mechanisms of protection, respectively).These categories of response have been termed Th1-type responses(cell-mediated response), and Th2-type immune responses (humoralresponse).

Stimulation of an immune response can result from a direct or indirectresponse of a cell or component of the immune system to an intervention(e.g., exposure to an immunogen). Immune responses can be measured inmany ways including activation, proliferation or differentiation ofcells of the immune system (e.g., B cells, T cells, dendritic cells,APCs, macrophages, NK cells, NKT cells etc.); up-regulated ordown-regulated expression of markers and cytokines; stimulation of IgA,IgM, or IgG titer; splenomegaly (including increased spleencellularity); hyperplasia and mixed cellular infiltrates in variousorgans. Other responses, cells, and components of the immune system thatcan be assessed with respect to immune stimulation are known in the art.

Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, compositions andmethods of the present invention induce expression and secretion ofcytokines (e.g., by macrophages, dendritic cells and CD4+ T cells).Modulation of expression of a particular cytokine can occur locally orsystemically. It is known that cytokine profiles can determine T cellregulatory and effector functions in immune responses. In someembodiments, Th1-type cytokines can be induced, and thus, theimmunostimulatory compositions of the present invention can promote aTh1 type antigen-specific immune response including cytotoxic T-cells(e.g., thereby avoiding unwanted Th2 type immune responses (e.g.,generation of Th2 type cytokines (e.g., IL-13) involved in enhancing theseverity of disease (e.g., IL-13 induction of mucus formation))).

Cytokines play a role in directing the T cell response. Helper (CD4+) Tcells orchestrate the immune response of mammals through production ofsoluble factors that act on other immune system cells, including B andother T cells. Most mature CD4+ T helper cells express one of twocytokine profiles: Th1 or Th2. Th1-type CD4+ T cells secrete IL-2, IL-3,IFN-γ, GM-CSF and high levels of TNF-α. Th2 cells express IL-3, IL-4,IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-α. Th1 typecytokines promote both cell-mediated immunity, and humoral immunity thatis characterized by immunoglobulin class switching to IgG2a in mice andIgG1 in humans. Th1 responses may also be associated with delayed-typehypersensitivity and autoimmune disease. Th2 type cytokines induceprimarily humoral immunity and induce class switching to IgG1 and IgE.The antibody isotypes associated with Th1 responses generally haveneutralizing and opsonizing capabilities whereas those associated withTh2 responses are associated more with allergic responses.

Several factors have been shown to influence skewing of an immuneresponse towards either a Th1 or Th2 type response. The bestcharacterized regulators are cytokines. IL-12 and IFN-γ are positive Th1and negative Th2 regulators. IL-12 promotes IFN-γ production, and IFN-γprovides positive feedback for IL-12. IL-4 and IL-10 appear importantfor the establishment of the Th2 cytokine profile and to down-regulateTh1 cytokine production.

Thus, in preferred embodiments, the present invention provides a methodof stimulating a Th1-type immune response in a subject comprisingadministering to a subject a composition comprising an immunogen.However, in other embodiments, the present invention provides a methodof stimulating a Th2-type immune response in a subject (e.g., ifbalancing of a T cell mediated response is desired) comprisingadministering to a subject a composition comprising an immunogen. Infurther preferred embodiments, adjuvants can be used (e.g., can beco-administered with a composition of the present invention) to skew animmune response toward either a Th1 or Th2 type immune response. Forexample, adjuvants that induce Th2 or weak Th1 responses include, butare not limited to, alum, saponins, and SB-As4. Adjuvants that induceTh1 responses include but are not limited to MPL, MDP, ISCOMS, IL-12,IFN-γ, and SB-AS2.

Several other types of Th1-type immunogens can be used (e.g., as anadjuvant) in compositions and methods of the present invention. Theseinclude, but are not limited to, the following. In some embodiments,monophosphoryl lipid A (e.g., in particular 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL)), is used. 3D-MPL is a well knownadjuvant manufactured by Ribi Immunochem, Montana. Chemically it isoften supplied as a mixture of 3-de-O-acylated monophosphoryl lipid Awith either 4, 5, or 6 acylated chains. In some embodiments,diphosphoryl lipid A, and 3-O-deacylated variants thereof are used. Eachof these immunogens can be purified and prepared by methods described inGB 2122204B, hereby incorporated by reference in its entirety. Otherpurified and synthetic lipopolysaccharides have been described (See,e.g., U.S. Pat. No. 6,005,099 and EP 0 729 473; Hilgers et al., 1986,Int. Arch. Allergy. Immunol., 79(4):392-6; Hilgers et al., 1987,Immunology, 60(1):141-6; and EP 0 549 074, each of which is herebyincorporated by reference in its entirety). In some embodiments, 3D-MPLis used in the form of a particulate formulation (e.g., having a smallparticle size less than 0.2 μm in diameter, described in EP 0 689 454,hereby incorporated by reference in its entirety).

In some embodiments, saponins are used as an immunogen (e.g., Th1-typeadjuvant) in a composition of the present invention. Saponins are wellknown adjuvants (See, e.g., Lacaille-Dubois and Wagner (1996)Phytomedicine vol 2 pp 363-386). Examples of saponins include Quil A(derived from the bark of the South American tree Quillaja SaponariaMolina), and fractions thereof (See, e.g., U.S. Pat. No. 5,057,540;Kensil, Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0362 279, each of which is hereby incorporated by reference in itsentirety). Also contemplated to be useful in the present invention arethe haemolytic saponins QS7, QS17, and QS21 (HPLC purified fractions ofQuil A; See, e.g., Kensil et al. (1991). J. Immunology 146, 431-437,U.S. Pat. No. 5,057,540; WO 96/33739; WO 96/11711 and EP 0 362 279, eachof which is hereby incorporated by reference in its entirety). Alsocontemplated to be useful are combinations of QS21 and polysorbate orcyclodextrin (See, e.g., WO 99/10008, hereby incorporated by referencein its entirety.

In some embodiments, an immunogenic oligonucleotide containingunmethylated CpG dinucleotides (“CpG”) is used as an adjuvant. CpG is anabbreviation for cytosine-guanosine dinucleotide motifs present in DNA.CpG is known in the art as being an adjuvant when administered by bothsystemic and mucosal routes (See, e.g., WO 96/02555, EP 468520, Davis etal., J. Immunol, 1998, 160(2):870-876; McCluskie and Davis, J. Immunol.,1998, 161(9):4463-6; and U.S. Pat. App. No. 20050238660, each of whichis hereby incorporated by reference in its entirety). For example, insome embodiments, the immunostimulatory sequence isPurine-Purine-C-G-pyrimidine-pyrimidine; wherein the CG motif is notmethylated.

Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, the presence of oneor more CpG oligonucleotides activate various immune subsets includingnatural killer cells (which produce IFN-γ) and macrophages. In someembodiments, CpG oligonucleotides are formulated into a composition ofthe present invention for inducing an immune response. In someembodiments, a free solution of CpG is co-administered together with anantigen (e.g., present within a solution (See, e.g., WO 96/02555; herebyincorporated by reference). In some embodiments, a CpG oligonucleotideis covalently conjugated to an antigen (See, e.g., WO 98/16247, herebyincorporated by reference), or formulated with a carrier such asaluminium hydroxide (See, e.g., Brazolot-Millan et al., Proc. Natl. AcadSci., USA, 1998, 95(26), 15553-8).

In some embodiments, adjuvants such as Complete Freunds Adjuvant andIncomplete Freunds Adjuvant, cytokines (e.g., interleukins (e.g., IL-2,IFN-γ, IL-4, etc.), macrophage colony stimulating factor, tumor necrosisfactor, etc.), detoxified mutants of a bacterial ADP-ribosylating toxinsuch as a cholera toxin (CT), a pertussis toxin (PT), or an E. Coliheat-labile toxin (LT), particularly LT-K63 (where lysine is substitutedfor the wild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S109 (whereserine is substituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (See, e.g.,WO93/13202 and WO92/19265, each of which is hereby incorporated byreference), and other immunogenic substances (e.g., that enhance theeffectiveness of a composition of the present invention) are used with acomposition comprising an immunogen of the present invention.

Additional examples of adjuvants that find use in the present inventioninclude poly(dicarboxylatophenoxy) phosphazene (PCPP polymer; VirusResearch Institute, USA); derivatives of lipopolysaccharides such asmonophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton,Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide(t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OMPharma SA, Meyrin, Switzerland); and Leishmania elongation factor (apurified Leishmania protein; Corixa Corporation, Seattle, Wash.).

Adjuvants may be added to a composition comprising an immunogen, or, theadjuvant may be formulated with carriers, for example liposomes, ormetallic salts (e.g., aluminium salts (e.g., aluminium hydroxide)) priorto combining with or co-administration with a composition.

In some embodiments, a composition comprising an immunogen comprises asingle adjuvant. In other embodiments, a composition comprises two ormore adjuvants (See, e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO98/56414; WO 99/12565; WO 99/11241; and WO 94/00153, each of which ishereby incorporated by reference in its entirety).

In some embodiments, a composition comprising an immunogen comprises oneor more mucoadhesives (See, e.g., U.S. Pat. App. No. 20050281843, herebyincorporated by reference in its entirety). The present invention is notlimited by the type of mucoadhesive utilized. Indeed, a variety ofmucoadhesives are contemplated to be useful in the present inventionincluding, but not limited to, cross-linked derivatives of poly(acrylicacid) (e.g., carbopol and polycarbophil), polyvinyl alcohol, polyvinylpyrollidone, polysaccharides (e.g., alginate and chitosan),hydroxypropyl methylcellulose, lectins, fimbrial proteins, andcarboxymethylcellulose. Although an understanding of the mechanism isnot necessary to practice the present invention and the presentinvention is not limited to any particular mechanism of action, in someembodiments, use of a mucoadhesive (e.g., in a composition comprising animmunogen) enhances induction of an immune response in a subject (e.g.,administered a composition of the present invention) due to an increasein duration and/or amount of exposure to an immunogen that a subjectexperiences when a mucoadhesive is used compared to the duration and/oramount of exposure to an immunogen in the absence of using themucoadhesive.

In some embodiments, a composition of the present invention may comprisesterile aqueous preparations. Acceptable vehicles and solvents include,but are not limited to, water, Ringer's solution, phosphate bufferedsaline and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed mineral or non-mineral oil maybe employed including synthetic mono-ordi-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.Carrier formulations suitable for mucosal, subcutaneous, intramuscular,intraperitoneal, intravenous, or administration via other routes may befound in Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa.

A composition comprising an immunogen of the present invention can beused therapeutically (e.g., to enhance an immune response) or as aprophylactic (e.g., for immunization (e.g., to prevent signs or symptomsof disease)). A composition comprising an immunogen of the presentinvention can be administered to a subject via a number of differentdelivery routes and methods.

For example, the compositions of the present invention can beadministered to a subject (e.g., mucosally (e.g., nasal mucosa, vaginalmucosa, etc.)) by multiple methods, including, but not limited to: beingsuspended in a solution and applied to a surface; being suspended in asolution and sprayed onto a surface using a spray applicator; beingmixed with a mucoadhesive and applied (e.g., sprayed or wiped) onto asurface (e.g., mucosal surface); being placed on or impregnated onto anasal and/or vaginal applicator and applied; being applied by acontrolled-release mechanism; being applied as a liposome; or beingapplied on a polymer.

In some embodiments, compositions of the present invention areadministered mucosally (e.g., using standard techniques; See, e.g.,Remington: The Science and Practice of Pharmacy, Mack PublishingCompany, Easton, Pa., 19th edition, 1995 (e.g., for mucosal deliverytechniques, including intranasal, pulmonary, vaginal and rectaltechniques), as well as European Publication No. 517,565 and Illum etal., J. Controlled Rel., 1994, 29:133-141 (e.g., for techniques ofintranasal administration), each of which is hereby incorporated byreference in its entirety). Alternatively, the compositions of thepresent invention may be administered dermally or transdermally, usingstandard techniques (See, e.g., Remington: The Science arid Practice ofPharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995). Thepresent invention is not limited by the route of administration.

Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, mucosal vaccinationis the preferred route of administration as it has been shown thatmucosal administration of antigens has a greater efficacy of inducingprotective immune responses at mucosal surfaces (e.g., mucosalimmunity), the route of entry of many pathogens. In addition, mucosalvaccination, such as intranasal vaccination, may induce mucosal immunitynot only in the nasal mucosa, but also in distant mucosal sites such asthe genital mucosa (See, e.g., Mestecky, Journal of Clinical Immunology,7:265-276, 1987). More advantageously, in further preferred embodiments,in addition to inducing mucosal immune responses, mucosal vaccinationalso induces systemic immunity. In some embodiments, non-parenteraladministration (e.g., muscosal administration of vaccines) provides anefficient and convenient way to boost systemic immunity (e.g., inducedby parenteral or mucosal vaccination (e.g., in cases where multipleboosts are used to sustain a vigorous systemic immunity)).

In some embodiments, a composition comprising an immunogen of thepresent invention may be used to protect or treat a subject susceptibleto, or suffering from, disease by means of administering a compositionof the present invention via a mucosal route (e.g., an oral/alimentaryor nasal route). Alternative mucosal routes include intravaginal andintra-rectal routes. In preferred embodiments of the present invention,a nasal route of administration is used, termed “intranasaladministration” or “intranasal vaccination” herein. Methods ofintranasal vaccination are well known in the art, including theadministration of a droplet or spray form of the vaccine into thenasopharynx of a subject to be immunized. In some embodiments, anebulized or aerosolized composition is provided. Enteric formulationssuch as gastro resistant capsules for oral administration, suppositoriesfor rectal or vaginal administration also form part of this invention.Compositions of the present invention may also be administered via theoral route. Under these circumstances, a composition comprising animmunogen may comprise a pharmaceutically acceptable excipient and/orinclude alkaline buffers, or enteric capsules. Formulations for nasaldelivery may include those with dextran or cyclodextran and saponin asan adjuvant.

Compositions of the present invention may also be administered via avaginal route. In such cases, a composition comprising an immunogen maycomprise pharmaceutically acceptable excipients and/or emulsifiers,polymers (e.g., CARBOPOL), and other known stabilizers of vaginal creamsand suppositories. In some embodiments, compositions of the presentinvention are administered via a rectal route. In such cases,compositions may comprise excipients and/or waxes and polymers known inthe art for forming rectal suppositories.

In some embodiments, the same route of administration (e.g., mucosaladministration) is chosen for both a priming and boosting vaccination.In some embodiments, multiple routes of administration are utilized(e.g., at the same time, or, alternatively, sequentially) in order tostimulate an immune response.

For example, in some embodiments, a composition comprising an immunogenis administered to a mucosal surface of a subject in either a priming orboosting vaccination regime. Alternatively, in some embodiments, thecomposition is administered systemically in either a priming or boostingvaccination regime. In some embodiments, a composition comprising animmunogen is administered to a subject in a priming vaccination regimenvia mucosal administration and a boosting regimen via systemicadministration. In some embodiments, a composition comprising animmunogen is administered to a subject in a priming vaccination regimenvia systemic administration and a boosting regimen via mucosaladministration. Examples of systemic routes of administration include,but are not limited to, a parenteral, intramuscular, intradermal,transdermal, subcutaneous, intraperitoneal or intravenousadministration. A composition comprising an immunogen may be used forboth prophylactic and therapeutic purposes.

In some embodiments, compositions of the present invention areadministered by pulmonary delivery. For example, a composition of thepresent invention can be delivered to the lungs of a subject (e.g., ahuman) via inhalation (e.g., thereby traversing across the lungepithelial lining to the blood stream (See, e.g., Adjei, et al.Pharmaceutical Research 1990; 7:565-569; Adjei, et al. Int. J.Pharmaceutics 1990; 63:135-144; Braquet, et al. J. CardiovascularPharmacology 1989 143-146; Hubbard, et al. (1989) Annals of InternalMedicine, Vol. III, pp. 206-212; Smith, et al. J. Clin. Invest. 1989;84:1145-1146; Oswein, et al. “Aerosolization of Proteins”, 1990;Proceedings of Symposium on Respiratory Drug Delivery II Keystone,Colorado; Debs, et al. J. Immunol. 1988; 140:3482-3488; and U.S. Pat.No. 5,284,656 to Platz, et al, each of which are hereby incorporated byreference in its entirety). A method and composition for pulmonarydelivery of drugs for systemic effect is described in U.S. Pat. No.5,451,569 to Wong, et al., hereby incorporated by reference; See alsoU.S. Pat. No. 6,651,655 to Licalsi et al., hereby incorporated byreference in its entirety)).

Further contemplated for use in the practice of this invention are awide range of mechanical devices designed for pulmonary and/or nasalmucosal delivery of pharmaceutical agents including, but not limited to,nebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices suitable for the practice of thisinvention are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis,Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood,Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research TrianglePark, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford,Mass.). All such devices require the use of formulations suitable fordispensing of the therapeutic agent. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to the usual diluents,adjuvants, surfactants, carriers and/or other agents useful in therapy.Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Thus, in some embodiments, a composition comprising an immunogen of thepresent invention may be used to protect and/or treat a subjectsusceptible to, or suffering from, a disease by means of administeringthe composition by mucosal, intramuscular, intraperitoneal, intradermal,transdermal, pulmonary, intravenous, subcutaneous or other route ofadministration described herein. Methods of systemic administration ofthe vaccine preparations may include conventional syringes and needles,or devices designed for ballistic delivery of solid vaccines (See, e.g.,WO 99/27961, hereby incorporated by reference), or needleless pressureliquid jet device (See, e.g., U.S. Pat. Nos. 4,596,556; 5,993,412, eachof which are hereby incorporated by reference), or transdermal patches(See, e.g., WO 97/48440; WO 98/28037, each of which are herebyincorporated by reference). The present invention may also be used toenhance the immunogenicity of antigens applied to the skin (transdermalor transcutaneous delivery, See, e.g., WO 98/20734; WO 98/28037, each ofwhich are hereby incorporated by reference). Thus, in some embodiments,the present invention provides a delivery device for systemicadministration, pre-filled with the vaccine composition of the presentinvention.

The present invention is not limited by the type of subject administered(e.g., in order to stimulate an immune response (e.g., in order togenerate protective immunity (e.g., mucosal and/or systemic immunity)))a composition of the present invention. Indeed, a wide variety ofsubjects are contemplated to be benefited from administration of acomposition of the present invention. In preferred embodiments, thesubject is a human. In some embodiments, human subjects are of any age(e.g., adults, children, infants, etc.) that have been or are likely tobecome exposed to a microorganism (e.g., E. coli). In some embodiments,the human subjects are subjects that are more likely to receive a directexposure to pathogenic microorganisms or that are more likely to displaysigns and symptoms of disease after exposure to a pathogen (e.g., immunesuppressed subjects). In some embodiments, the general public isadministered (e.g., vaccinated with) a composition of the presentinvention (e.g., to prevent the occurrence or spread of disease). Forexample, in some embodiments, compositions and methods of the presentinvention are utilized to vaccinate a group of people (e.g., apopulation of a region, city, state and/or country) for their own health(e.g., to prevent or treat disease). In some embodiments, the subjectsare non-human mammals (e.g., pigs, cattle, goats, horses, sheep, orother livestock; or mice, rats, rabbits or other animal). In someembodiments, compositions and methods of the present invention areutilized in research settings (e.g., with research animals).

A composition of the present invention may be formulated foradministration by any route, such as mucosal, oral, transdermal,intranasal, parenteral or other route described herein. The compositionsmay be in any one or more different forms including, but not limited to,tablets, capsules, powders, granules, lozenges, foams, creams or liquidpreparations.

Topical formulations of the present invention may be presented as, forinstance, ointments, creams or lotions, foams, and aerosols, and maycontain appropriate conventional additives such as preservatives,solvents (e.g., to assist penetration), and emollients in ointments andcreams.

Topical formulations may also include agents that enhance penetration ofthe active ingredients through the skin. Exemplary agents include abinary combination of N-(hydroxyethyl) pyrrolidone and a cell-envelopedisordering compound, a sugar ester in combination with a sulfoxide orphosphine oxide, and sucrose monooleate, decyl methyl sulfoxide, andalcohol.

Other exemplary materials that increase skin penetration includesurfactants or wetting agents including, but not limited to,polyoxyethylene sorbitan mono-oleoate (Polysorbate 80); sorbitanmono-oleate (Span 80); p-isooctyl polyoxyethylene-phenol polymer (TritonWR-1330); polyoxyethylene sorbitan tri-oleate (Tween 85); dioctyl sodiumsulfosuccinate; and sodium sarcosinate (Sarcosyl NL-97); and otherpharmaceutically acceptable surfactants.

In certain embodiments of the invention, compositions may furthercomprise one or more alcohols, zinc-containing compounds, emollients,humectants, thickening and/or gelling agents, neutralizing agents, andsurfactants. Water used in the formulations is preferably deionizedwater having a neutral pH. Additional additives in the topicalformulations include, but are not limited to, silicone fluids, dyes,fragrances, pH adjusters, and vitamins. Topical formulations may alsocontain compatible conventional carriers, such as cream or ointmentbases and ethanol or oleyl alcohol for lotions. Such carriers may bepresent as from about 1% up to about 98% of the formulation. Theointment base can comprise one or more of petrolatum, mineral oil,ceresin, lanolin alcohol, panthenol, glycerin, bisabolol, cocoa butterand the like.

In some embodiments, pharmaceutical compositions of the presentinvention may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The compositions of the present invention mayadditionally contain other adjunct components conventionally found inpharmaceutical compositions. Thus, for example, the compositions maycontain additional, compatible, pharmaceutically-active materials suchas, for example, antipruritics, astringents, local anesthetics oranti-inflammatory agents, or may contain additional materials useful inphysically formulating various dosage forms of the compositions of thepresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, preferably do not unduly interfere with thebiological activities of the components of the compositions of thepresent invention. The formulations can be sterilized and, if desired,mixed with auxiliary agents (e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, colorings, flavorings and/or aromatic substances andthe like) that do not deleteriously interact with the immunogen or othercomponents of the formulation. In some embodiments, immunostimulatorycompositions of the present invention are administered in the form of apharmaceutically acceptable salt. When used the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically acceptable saltsthereof. Such salts include, but are not limited to, those prepared fromthe following acids: hydrochloric, hydrobromic, sulphuric, nitric,phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric,citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include, but are not limited to, acetic acidand a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid anda salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).Suitable preservatives may include benzalkonium chloride (0.003-0.03%w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) andthimerosal (0.004-0.02% w/v).

In some embodiments, vaccine compositions are co-administered with oneor more other agents useful in treating cancer or infection by HCV.There are an enormous amount of antimicrobial agents currently availablefor use in treating bacterial, fungal and viral infections. For acomprehensive treatise on the general classes of such drugs and theirmechanisms of action, the skilled artisan is referred to Goodman &Gilman's “The Pharmacological Basis of Therapeutics” Eds. Hardman etal., 9th Edition, Pub. McGraw Hill, chapters 43 through 50, 1996,(herein incorporated by reference in its entirety). Generally, theseagents include agents that inhibit cell wall synthesis (e.g.,penicillins, cephalosporins, cycloserine, vancomycin, bacitracin); andthe imidazole antifungal agents (e.g., miconazole, ketoconazole andclotrimazole); agents that act directly to disrupt the cell membrane ofthe microorganism (e.g., detergents such as polmyxin and colistimethateand the antifungals nystatin and amphotericin B); agents that affect theribosomal subunits to inhibit protein synthesis (e.g., chloramphenicol,the tetracyclines, erthromycin and clindamycin); agents that alterprotein synthesis and lead to cell death (e g, aminoglycosides); agentsthat affect nucleic acid metabolism (e.g., the rifamycins and thequinolones); the antimetabolites (e.g., trimethoprim and sulfonamides);and the nucleic acid analogues such as zidovudine, gangcyclovir,vidarabine, and acyclovir which act to inhibit viral enzymes essentialfor DNA synthesis. Various combinations of antimicrobials may beemployed.

The present invention also includes methods involving co-administrationof a vaccine composition comprising an immunogen with one or moreadditional active and/or immunostimulatory agents (e.g., a compositioncomprising a different immunogen, an antibiotic, anti-oxidant, etc.).Indeed, it is a further aspect of this invention to provide methods forenhancing prior art immunostimulatory methods (e.g., immunizationmethods) and/or pharmaceutical compositions by co-administering acomposition of the present invention. In co-administration procedures,the agents may be administered concurrently or sequentially. In oneembodiment, the compositions described herein are administered prior tothe other active agent(s). The pharmaceutical formulations and modes ofadministration may be any of those described herein. In addition, thetwo or more co-administered agents may each be administered usingdifferent modes (e.g., routes) or different formulations. The additionalagents to be co-administered (e.g., antibiotics, adjuvants, etc.) can beany of the well-known agents in the art, including, but not limited to,those that are currently in clinical use.

In some embodiments, a composition comprising an immunogen isadministered to a subject via more than one route. For example, asubject that would benefit from having a protective immune response (eg, immunity) towards a pathogenic microorganism may benefit fromreceiving mucosal administration (e.g., nasal administration or othermucosal routes described herein) and, additionally, receiving one ormore other routes of administration (e.g., parenteral or pulmonaryadministration (e.g., via a nebulizer, inhaler, or other methodsdescribed herein). In some preferred embodiments, administration viamucosal route is sufficient to induce both mucosal as well as systemicimmunity towards an immunogen or organism from which the immunogen isderived. In other embodiments, administration via multiple routes servesto provide both mucosal and systemic immunity. Thus, although anunderstanding of the mechanism is not necessary to practice the presentinvention and the present invention is not limited to any particularmechanism of action, in some embodiments, it is contemplated that asubject administered a composition of the present invention via multipleroutes of administration (e.g., immunization (e.g., mucosal as well asairway or parenteral administration of the composition) may have astronger immune response to an immunogen than a subject administered acomposition via just one route.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions, increasing convenience to thesubject and a physician. Many types of release delivery systems areavailable and known to those of ordinary skill in the art. They includepolymer based systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109,hereby incorporated by reference. Delivery systems also includenon-polymer systems that are: lipids including sterols such ascholesterol, cholesterol esters and fatty acids or neutral fats such asmono-di- and tri-glycerides; hydrogel release systems; sylastic systems;peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which an agent of the invention is contained in a form withina matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189,and 5,736,152, each of which is hereby incorporated by reference and (b)diffusional systems in which an active component permeates at acontrolled rate from a polymer such as described in U.S. Pat. Nos.3,854,480, 5,133,974 and 5,407,686, each of which is hereby incorporatedby reference. In addition, pump-based hardware delivery systems can beused, some of which are adapted for implantation.

In some embodiments, a vaccine composition of the present invention isformulated in a concentrated dose that can be diluted prior toadministration to a subject. For example, dilutions of a concentratedcomposition may be administered to a subject such that the subjectreceives any one or more of the specific dosages provided herein. Insome embodiments, dilution of a concentrated composition may be madesuch that a subject is administered (e.g., in a single dose) acomposition comprising 0.5-50% of a nanemulsion and immunogen present inthe concentrated composition. Concentrated compositions are contemplatedto be useful in a setting in which large numbers of subjects may beadministered a composition of the present invention (e.g., animmunization clinic, hospital, school, etc.). In some embodiments, acomposition comprising an immunogen of the present invention (e.g., aconcentrated composition) is stable at room temperature for more than 1week, in some embodiments for more than 2 weeks, in some embodiments formore than 3 weeks, in some embodiments for more than 4 weeks, in someembodiments for more than 5 weeks, and in some embodiments for more than6 weeks.

In some embodiments, following an initial administration of acomposition of the present invention (e.g., an initial vaccination), asubject may receive one or more boost administrations (e.g., around 2weeks, around 3 weeks, around 4 weeks, around 5 weeks, around 6 weeks,around 7 weeks, around 8 weeks, around 10 weeks, around 3 months, around4 months, around 6 months, around 9 months, around 1 year, around 2years, around 3 years, around 5 years, around 10 years) subsequent to afirst, second, third, fourth, fifth, sixth, seventh, eights, ninth,tenth, and/or more than tenth administration. Although an understandingof the mechanism is not necessary to practice the present invention andthe present invention is not limited to any particular mechanism ofaction, in some embodiments, reintroduction of an immunogen in a boostdose enables vigorous systemic immunity in a subject. The boost can bewith the same formulation given for the primary immune response, or canbe with a different formulation that contains the immunogen. The dosageregimen will also, at least in part, be determined by the need of thesubject and be dependent on the judgment of a practitioner.

Dosage units may be proportionately increased or decreased based onseveral factors including, but not limited to, the weight, age, andhealth status of the subject. In addition, dosage units may be increasedor decreased for subsequent administrations (e.g., boostadministrations).

It is contemplated that the compositions and methods of the presentinvention will find use in various settings, including researchsettings. For example, compositions and methods of the present inventionalso find use in studies of the immune system (e.g., characterization ofadaptive immune responses (e.g., protective immune responses (e.g.,mucosal or systemic immunity))). Uses of the compositions and methodsprovided by the present invention encompass human and non-human subjectsand samples from those subjects, and also encompass researchapplications using these subjects. Compositions and methods of thepresent invention are also useful in studying and optimizingnanoemulsions, immunogens, and other components and for screening fornew components. Thus, it is not intended that the present invention belimited to any particular subject and/or application setting.

The present invention further provides kits comprising the vaccinecompositions comprised herein. In some embodiments, the kit includes allof the components necessary, sufficient or useful for administering thevaccine. For example, in some embodiments, the kits comprise devices foradministering the vaccine (e.g., needles or other injection devices),temperature control components (e.g., refrigeration or other coolingcomponents), sanitation components (e.g., alcohol swabs for sanitizingthe site of injection) and instructions for administering the vaccine.

In some embodiments, the present invention provides a platform (e.g.,systems and methods) for identifying CTL epitopes. Embodiments of thepresent invention utilize T cell repertoires that have not been renderedtolerant as tools for efficient and sensitive detection of the epitopes.More precisely, for discovery of epitopes from self-TAA by non-tolerantT cells, the self-TAA are presented on HLA-molecules for which the Tcell donor is negative. Thus, the self-TAA are presented onantigen-presenting cells expressing HLA-A2, whereas the T cells areHLA-A2 negative. T cells recognizing peptides presented on foreign HLAare termed allo-reactive, or allo-restricted.

In some embodiments, the method comprises the following steps: (1)Candidate target proteins are screened for cell-type specific expressionin normal and malignant hematopoietic cells (GeneSapiens.org). (2)Selected target proteins, and HLA-A*02:01, are cloned and recombinedinto a vector for in vitro production of mRNA encoding full-lengthprotein. (3) Monocyte-derived dendritic cells from HLA-A2neg donors aretransfected with the mRNA, and subsequently co-cultured with autologous,non-adherent peripheral blood mononuclear cells. The cultures may bere-stimulated with EBV-LCLs transfected with mRNA encoding the targetprotein and HLA-A2 on days 12, and 19 (not shown, and optional). (4)Selected targets are subjected to affinity-based algorithmic predictionof HLA-A*02:01-restricted peptides (cbs.dtu.dk/services/NetMHC/) andthose predicted to bind with the highest affinities are synthesized. (5)These peptides are complexed with HLA-A*02:01 monomers by UV-inducedligand exchange, followed by multimerization using streptavidin (SA)conjugated to, e.g., PE, APC or PerCP-Cy5.5, respectively. (6) By end ofculture (e.g. days 12, 19 or 26), CD8 positive T cells reactive todifferent epitopes are detected by flow cytometric measurements ofcombinations of fluorescently labeled pHLA-A2 multimers.

For discovery of epitopes from foreign antigens, for example Hepatitis CVirus (HCV), donors that are seronegative for HCV and have not undergoneinfection with the virus, are used, as the T cell repertoires from suchdonors are not rendered tolerant to the virus. For discovery of epitopesin foreign antigens, for instance proteins derived from Hepatitis CVirus, dendritic cells and T cells are harvested from the same HLA-A2positive donor, otherwise the procedure is the same as described above.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Materials and Methods

Healthy Donors and Cell Lines

This study was approved by the Regional Ethics Committee and performedin accordance with the declaration of Helsinki. Healthy donors wereHLA-typed by standard molecular techniques.

THP-1 and JVM-2 cells were obtained from Deutsche Sammlung vonMikroorganismen and Zellkulturen. The following cell lines were kindgifts: SupT1 cells from M. Pule (University College London, UK); K562(CCL 243) cell line from John Torgils Vaage; primary adiposetissue-derived mesenchymal stem cells (MSC) from J. Brinchmann'slaboratory (Institute of Immunology, Oslo University Hospital (OUS)Rikshospitalet); HaCaT keratinocyte cell line from Dr. F. Jahnsen (Deptof Pathology, OUS Rikshospitalet); colorectal carcinoma cell lineHCT-116 (CCL-247), colorectal adenocarcinoma cell line Caco-2 (HTB-37),hepatocellular carcinoma cell line Hep G2 (HB-8065) and lungadenocarcinoma cell line NCI-H522 (CRL-5810) from Ragnhild A. Lothe andGuro E. Lind (Dept of Cancer Prevention); FM81 and T2 cells from ElseMarit Inderberg Suso (Dept of Immunology) and HeLa cells from AndreasBrech (Dept of Biochemistry), all at OUS Radiumhospitalet. Platinum-E(Plat-E) retroviral packaging cell line (referred to as HEK) (CellBiolabs, San Diego, Calif., USA) and HeLa cells were maintained in DMEM(Invitrogen); MSC, HaCaT and HCT-116 were grown in D-MEM/F12 (Sigma);Caco-2 and Hep G2 in EMEM (ATCC®), all with 10% FCS andpenicillin/streptomycin (P/S). The other cell lines were cultured inRPMI 1640 (Gibco), with 10% FCS and P/S.

Prediction of HLA-A*02:01 Specific Peptides from Target Proteins

Protein sequences were obtained from NCBI Entrez engine. Prediction ofHLA-A*02:01-binding peptides from target proteins was performed usingthe algorithms NetMHCpan 2.2 (6) for MPO, or NetMHC 3.2 (40) for CD20and HCV. Peptides with an IC50 score of <500 nM were consideredpotential HLA-A2 binders, and are listed in Table 1.

Generation of Peptide-HLA Complexes

All predicted peptides, and control peptides ELAGIGILTV (SEQ ID NO:73)(MART-1) and NLVPMVATV (SEQ ID NO:75 ) (CMV), were synthesized byProimmune Ltd. (Oxford, UK) or GenScript Inc. (USA). Synthesis ofconditional HLA-A2 ligands containing a UV-sensitive peptide wasperformed as described in (13, 41). For generation of pMHC multimers,fluorochrome-streptavidin (SA) conjugates were generated as described in(13), with phycoerythrin (PE), allophycocyanin (APC) (BD Biosciences),or Peridinin-chlorophyll protein-Cy5.5 (PerCP-Cy5.5) (Ebiosciences).HLA-B*08:01 and HLA-B*07:02-restricted pentamers were from ProImmuneLtd. (Oxford, UK).

Plasmid Constructs, mRNA/DNA-Transfection and Retroviral Transduction

MPO was amplified from reverse-transcribed mRNA from HL-60 cells withPfx (Invitrogen) using the primers: 5′-caccATGGGGGTTCCCTTCTTC-3′ (SEQ IDNO:71) and 5′-TCAGGCGTAGTCGGGCACGTCGTAGGGGTAGGAGGCTTCCCTCCAGGAAG-3′ (SEQID NO:72), subcloned into pENTR/D-TOPO (Invitrogen) and recombined intothe Gateway compatible pCIpA102-G vector for mRNA production followingsequencing (42). The CD20 mRNA expression construct was reported in(23). The mRNA encoding HLA-A*02:01 or target proteins was generated asdescribed in (8), and used to transfect moDCs, EBV-LCL, HaCaT, or K562cells by electroporation (1250 V/cm for 3 ms (moDCs, HaCaT), 2 ms (K562)and 1 ms (EBV-LCL), respectively) with a BTX ECM 830 square waveelectroporator (BTX, Harvard Apparatus, Holliston, Mass., USA). HEK andHeLa cells were transfected with DNA encoding HLA-A2 and MPO or CD20using FuGENE-6 (Roche, Basel, Switzerland), following the manufacturer'sprotocol. Retroviral particles were produced and transduced as describedin (42). HCV core and NS3 genes were synthesized by GeneArt (Invitrogen)and inserted into a Gateway compatible vector (pENT221) and subsequentlyrecombined with pCIpA102-G. A Gateway compatible version of pMP71 wasused to express MPO and/or HLA-A*02:01 in SupT1, and the MART-1 (DMFS)TcR in primary T cells (construct derived from pMSGV-DMFS_2A, a kindgift from R. Morgan (NIH, Bethesda, USA)).

Induction of Multimer Reactive T Cells Restricted by Allogeneic HLA

Mature monocyte-derived dendritic cells (moDC) were generated asdescribed in (8) from HLA-A*02:01neg donors. One hour followingmRNA-transfection, cells were combined with thawed, autologousnon-adherent PBMC and cultured as described in (8, 23). Cells werere-stimulated day 12 and 19 with irradiated (25 Gy) HLA-A*02:01posEBV-LCL transfected 4 hrs earlier with mRNA encoding the target protein,in X-vivo 20 (BioWhittaker, Lonza, Walkersville, Md., USA) with 5%pooled human serum (Trina Bioreactives AG, Nänikon) and with IL-2supplemented at 10 U/ml on day 14 (R&D systems, USA).

Multimer Staining, Sorting and Expansion of Multimer Reactive CTL Lines

On day 26, co-cultures were stained with the succinimidyl ester dyePacific Blue (Invitrogen Molecular Probes, USA) for live/dead celldiscrimination at a final concentration of 0.1 ug/mL for 10 min at 37°C. in PBS. Two μl of 1-3 pHLA-A*02:01 tetramer(s) (in-house), orpentamers of HLA-B*07:02 or HLA-B*08:01 (Proimmune), were added for 15min at RT to stain 1×106 co-cultured cells, followed by anti-CD8.Sorting was performed using FACS Aria or FACS Vantage SE Cell sorters(BD Biosciences, San Jose, Calif., USA). Sorted, tetramer positive cellswere expanded in the presence of irradiated allogeneic PBMC in X-Vivo 20medium with 5% pooled human serum, IL-2 (50 U/ml, R&D systems, USA),IL-15 (2 ng/ml, Peprotech EC Ltd, USA) and PHA (1 μg/ml, Remel) for 3-4weeks.

Antibodies and Flow Cytometry

Cells were labelled with the following antibodies, in-house conjugatedto Alexa Fluor 647 or Atto 488: anti-CD8 (RPA-T8), -CD107a (H4A3),-CD107b (H4B4) (all from BD Biosciences), and -HLA-A2 (BB7.2; AbDSerotec), or -CD4FITC, -MPO-PE (MPO-7; Dako, Denmark), -CD14PerCP-Cy5.5,-IFN-γPE, CD8PerCP-Cy5.5 or CD8Pacific Blue (SKI, Ebiosciences, USA).Cells were analyzed on FACS LSR II and data analysis performed with FACSDiVa (both from BD Biosciences) or FlowJo softwares (Tree Star Inc.,Ashland, Oreg., USA). Functional T cell responses were measured asmobilization of CD107a/b (degranulation) and production of IFN-γ by flowcytometry, as described in (23), following co-culture with varioustarget cells that were DNA-transfected 48 h earlier, mRNA-transfected4-10 h earlier, or peptide-loaded (10 ug/ml for 4 h, followed bywashing). CD4pos T cells were isolated using a CD4 positive isolationkit (Dynal Biotech, Norway), whereas monocytes were isolated byelutriation centrifugation.

Results

Detection of 36 Novel Epitopes in the Tumor-Associated Antigens CD20 andMPO by High-Throughput Induction of Allo-Restricted CTLs

It was contemplated that T-cell repertoires that had not been renderedtolerant would allow efficient and sensitive assessment of theimmunopeptidome. This was tested in a setting where peptide presentationof self-antigens by foreign HLA was induced using mRNA transfection offull-length target proteins. Monocyte-derived DCs from HLA-A2neg donorswere transfected with HLA-A2, which we have demonstrated is an efficientstrategy for peptide presentation on allogeneic HLA (8). The moDCs wereco-transfected with mRNA encoding full-length CD20 or MPO, representingleukemia-associated antigens with an expression pattern confirmed to behighly restricted to normal and malignant hematopoietic cells across10,000 samples (genesapiens.org). Autologous CD8pos T cells that hadbeen co-cultured with engineered moDCs were labelled with a panel ofcolor-coded peptide-HLA-A2 (pHLA) multimers containing peptides fromCD20 or MPO that were predicted to bind HLA-A*02:01 (FIG. 1). A total of50 peptides, ranked as strong to intermediate binders (<500 nM) (TableS1), were synthesized and used to generate pHLA multimers for T cellstaining (FIG. 1A). pHLA multimer staining revealed that co-culture of Tcells with CD20-transfected moDCs resulted in generation of T cellsreactive with the majority of the 20 peptides from CD20 (FIG. 1B).Specificity of this interaction was shown by the fact that thesepeptides were not recognized by T cells primed by MPO-transfected DCs(FIG. 1B). Vice versa, T cells primed by MPO-transfected cellsrecognized the majority of the peptides from MPO (FIG. 1C), whereasthese MPO peptides were not recognized by the cells primed withCD20-transfected DCs (FIG. 1C). HLA multimers containing peptides fromirrelevant antigens (MART-1 and CMV) were not recognized by any of the Tcells in the two culture conditions (FIG. 1B-C, Ctrl).

In summary, the approach resulted in detection of 14/20 of the predictedCD20 epitopes, and 23/30 predicted MPO epitopes. Of the 37 identifiedepitopes, 36 were novel. The generated CTLs reacted only with relevantpHLA multimers and not with a large number of irrelevant peptides,indicating the specific induction of these reactivities exclusively inthe presence of the relevant antigen.

Allo-Restricted CTL Lines Recognize Peptides Presented on Leukemic andNon-Malignant Hematopoietic Cells Endogenously Expressing CD20 or MPO,and are Highly Peptide- and HLA Specific

Dendritic cells are the most effective antigen-presenting cells known.Thus, it was investigated whether transfected cells present peptidesthat are not found on HLA molecules on leukemic cells. To assess whetherthe epitope repertoire was equally broad on cells endogenouslyexpressing these target proteins, we randomly selected 13 out of the 37multimer-reactive CTL populations for sorting, expansion of CTL linesand functional testing. The CTL lines were tested for reactivity againsta panel of up to 15 HLA-A2pos target cells that were positive ornegative for CD20 and/or MPO, respectively.

The CTL lines reactive to four different peptides from CD20 respondedstrongly by degranulation and interferon (IFN)-γ production to HLA-A2postarget cells endogenously expressing CD20 (FIG. 2A,B). These includedEBV-transformed lymphoblastoid cell lines (EBV-LCLs), theB-prolymphocytic leukemia cell line JVM-2, but alsoCD20/HLA-A2-transfected HeLa cells (FIG. 2A,B). Reactivity was alsotested against a variety of cell lines of different histologies thatexpressed HLA-A2, but not CD20, such as colon carcinoma (HCT-116,Caco-2), liver carcinoma (Hep G2), lung adenocarcinoma (NCI-H522),keratinocytes (HaCaT), malignant melanoma (FM81), human embryonic kidney(HEK), cervix adenocarcinoma (HeLa), T cell lymphoblastic lymphoma(SupT1), chronic myelogeneous leukemia (K562) and mesenchymal stem cells(MSC) (FIG. 2B). No or only very low reactivity was seen against thiswide range of targets (FIG. 2A,B). However, responses against thesetarget cells were again observed when the cells were loaded externallywith the relevant peptide. Furthermore, the four CD20-reactive CTL linesdid not respond to the acute myelomonocytic leukemia cell line THP-1,unless loaded with relevant CD20 peptides (FIG. 5A).

Similarly, CTL lines reactive to 7 different peptides from MPO respondedstrongly by degranulation and IFN-γ production to HLA-A2pos target cellsthat endogenously express MPO; primary monocytes and the acutemyelomonocytic leukemia cell line THP-1. Furthermore, these CTL linesalso responded to MPO/HLA-A2-transfected HeLa cells. In contrast,negligible recognition was observed for the panel of HLA-A2pos antigennegative target cell lines, whereas responses in all cases again becameapparent when these target cells were loaded with the relevant MPOpeptide (FIG. 3).

CTL lines reactive to two additional MPO-derived peptides were generatedfrom the same donor and tested functionally against a smaller selectionof target cells in separate experiments. These CTL lines respondedstrongly to HLA-A2pos target cells endogenously expressing MPO, eithernaturally (monocytes, THP-1) or following genetic introduction (SupT1,HeLa), and to HLA-A2posMPOneg target cells loaded with relevant peptide(SupT1, T2, HEK, CD4pos T cells) (FIG. 5B,C). Negligible responses wereseen to the MPOneg target cells in the absence of added antigen.Furthermore, strong responses and a high degree of specificity wasconfirmed for additional cell lines generated against 5 of the sameMPO-derived peptides from a second donor (FIG. 5D, E).

To obtain more information on the range of T cell reactivities to one ofthese novel T cell epitopes, T-cell clones were generated from CTL line#25, specific for MPO epitope LIQPFMFRL (SEQ ID NO:45). All CTL clonesresponded strongly to HLA-A2pos monocytes, whereas the responses toMPO-transfected or peptide-loaded HEK cells was variable, although inall cases higher than that observed for untransfected HEK cells (FIG.6A). Sufficient cells were available for 3 of the clones to evaluatetheir fine specificity using HLA multimers containing peptides in whicheach of the amino acids in the MPO peptide #25 was individually replacedby an alanine residue. The results showed that the fine-specificitydiffered among the T cell clones, with the large majority of thesubstitutions leading to reduced multimer binding, although replacementsin position 4 and 9 gave an increased binding (FIG. 6B). The higherbinding seen with an alanine-replacement in position 4 (P506A) was,however, likely due to unspecific binding, as this particular multimerwas the only one that bound non-specifically to primary T cellstransduced with a MART-1 specific TCR (FIG. 6C).

The high avidity of MPO-specific clones was additionally confirmed withclones from cell line #9 (FIG. 9). The clones responded to target cellsloaded with peptide at concentrations down to 1 pM. Furthermore, theyresponded strongly to THP-1 cells endogenously presenting MPO (FIG. 9).

The strong HLA multimer binding and CTL responses that were induced tovarious target cells indicated high functional avidities. The CTL linesare, however, likely to consist of T cell clones displaying a widevariety of reactivities, as previously shown for allo-restricted T cellclones (9) and here for clones recognizing peptide #25. Nevertheless,when performing peptide titrations, reactivity was demonstrated at thelowest concentration (100 pM) for all but one of the tested CTL lines,indicating that for all, or nearly all, epitopes highly sensitive T cellpopulations can be obtained (FIG. 7).

In conclusion, we here demonstrate that a total of 18 CTL lines,reactive to 13 different peptides, strongly recognize their cognateantigens but do not show detectable cross reactivity against a large setof target cells that do not express this antigen. In addition,reactivity against 24 other CD20- and MPO-derived epitopes was observed,which was not pursued further here. Collectively, these resultsdemonstrate that the majority of the algorithm-predicted peptides forCD20 and MPO were presented on the surface of cells endogenouslyexpressing these proteins, thereby forming a broad repertoire ofpotential T cell targets. Moreover, the strong target reactivity andhigh degree of HLA- and peptide specificity indicates that suchallo-reactive CTLs or their TCRs could be useful in a therapeuticsetting.

TABLE 1 Peptides predicted to bind HLA-A*02:01 from CD20 Predictedaffinity Peptide # Position Sequence (nM)   1* 187 SLFLGILSV   9(SEQ ID NO: 1)  2 115 KMIMNSLSL  25 (SEQ ID NO: 2)  3 197 LIFAFFQEL  26(SEQ ID NO: 3)  4 55 QIMNGLFHI  27 (SEQ ID NO: 4)  5 93 YIISGSLLA  28(SEQ ID NO: 5)  6 86 PLWGGIMYI  29 (SEQ ID NO: 6)  7 122 SLFAAISGM  33(SEQ ID NO: 7)  8 69 LMIPAGIYA  34 (SEQ ID NO: 8)  9 117 IMNSLSLFA  36(SEQ ID NO: 9) 10 189 FLGILSVML  44 (SEQ ID NO: 10) 11 67 GLLMIPAGI  55(SEQ ID NO: 11) 12 153 FIRAHTPYI  64 (SEQ ID NO: 12) 13 192 ILSVMLIFA 67 (SEQ ID NO: 13) 14 130 MILSIMDIL 114 (SEQ ID NO: 14) 15  91IMYIISGSL 116 (SEQ ID NO: 15) 16 134 IMDILNIKI 154 (SEQ ID NO: 16) 17 56IMNGLFHIA 167 (SEQ ID NO: 17) 18 126 AISGMILSI 170 (SEQ ID NO: 18) 19129 GMILSIMDI 306 (SEQ ID NO: 19) 20 184 SIQSLFLGI 376 (SEQ ID NO: 20)*Bae J., et al. Clin Cancer Res. 2004; 10: 7043-7052

TABLE 2 Peptides predicted to bind HLA-A*02:01 from myeloperoxidasePredicted affinity Peptide # Position Sequence (nM)  1  27 KLLLALAGL  20(SEQ ID NO: 21)  2  31 ALAGLLAIL  31 (SEQ ID NO: 22)  3 117 YLHVALDLL 29 (SEQ ID NO: 23)  4 131 SLWRRPFNV  12 (SEQ ID NO: 24)  5 142VLTPAQLNV  41 (SEQ ID NO: 25)  6 570 RLFEQVMRI   9 (SEQ ID NO: 26)  7642 WMGGVSEPL  24 (SEQ ID NO: 27)  8  28 LLLALAGLL 144 (SEQ ID NO: 28) 9  30 LALAGLLAI 433 (SEQ ID NO: 29) 10  52 VLGEVDTSL  85(SEQ ID NO: 30) 11  64 SMEEAKQLV 484 (SEQ ID NO: 31) 12  97 LLSYFKQPV 51(SEQ ID NO: 32) 13 249 LMFMQWGQL 315 (SEQ ID NO: 33) 14 257 LLDHDLDFT460 (SEQ ID NO: 34) 15 328 QINALTSFV 324 (SEQ ID NO: 35) 16 340MVYGSEEPL 392 (SEQ ID NO: 36) 17 354 NMSNQLGLL 399 (SEQ ID NO: 37) 18368 FQDNGRALL 277 (SEQ ID NO: 38) 19 380 NLHDDPCLL 218 (SEQ ID NO: 39)20 424 RLATELKSL 227 (SEQ ID NO: 40) 21 447 KIVGAMVQI 258(SEQ ID NO: 41) 22 461 YLPLVLGPT 401 (SEQ ID NO: 42) 23 484 SVDPRIANV156 (SEQ ID NO: 43) 24 488 RIANVFTNA 469 (SEQ ID NO: 44) 25 503LIQPFMFRL 317 (SEQ ID NO: 45) 26 510 RLDNRYQPM 482 (SEQ ID NO: 46) 27528 RVFFASWRV  51 (SEQ ID NO: 47) 28 537 VLEGGIDPI 249 (SEQ ID NO: 48)29 548 GLMATPAKL  72 (SEQ ID NO: 49) 30 726 FVNCSTLPA  55(SEQ ID NO: 50)

The results in the present study indicate that the self-immunopeptidomeis more diverse than previously believed, and that a multitude ofself-epitopes can be targeted by T cells. A surprisingly high fraction(74%) of peptides predicted to bind HLA from self-TAA were efficientlypresented to CTLs, using the strategy presented here (FIG. 4). Thisresulted in the detection of 37 epitopes from one known (CD20) and onenovel (MPO) cancer target, representing leukemia-associateddifferentiation antigens. Among these, 36 epitopes were novel. Thisoutput was striking, considering that only 250 HLA class I CTL cancerepitopes have been identified to date from non-melanoma-associatedantigens (1). The epitopes identified in the present study werepresented on leukemia cells and other target cells endogenouslyexpressing the antigens, at levels sufficient to be efficientlyrecognized by T cells. These results indicate that algorithm-basedpredictions reflect the immunopeptidome more accurately than previouslybelieved. Furthermore, it suggests that studies performed using massspectrometry may underestimate MHC-associated peptide repertoires.Peptide ligands for HLA-A*02:01 have not been identified from the largemajority of the on average 18,000 proteins expressed in a cell (10). Inconcert with this, none of the novel epitopes from CD20 and MPO werefound in existing peptide databases, such as Syfpeithi (syfpeithi.de)and the Immune epitope database (iedb.org).

Our results have important implications for T-cell epitope discovery.Traditional cell-based methods typically involve cloning of patient Tcells, followed by laborious screening of clones for reactivity againstpeptide libraries or fractions of peptide pools eluted from MHCmolecules of target cells, and finally peptide characterization by MS(11, 12). Recently, color-coded pMHC-multimers (13) were used to screentumor-infiltrating lymphocytes for reactivity against hundreds of knownmelanoma epitopes, thereby increasing throughput (1). However, allstudies depending on patient cells for epitope discovery are limited bytolerance to self and previous immunization history of the individual.In contrast, the strategy presented here took advantage of induction ofresponses in T-cell repertoires from antigen-inexperienced,non-tolerized individuals. This suggests that the use of self-tolerantT-cell repertoires, rather than poor peptide presentation, is the reasonwhy few cancer epitopes have been identified. In the current approach,induction of T-cell responses by DCs transfected with mRNA encodingfull-length target proteins focused discovery on naturally presentedpeptides (14). Finally, detection of antigen-specific T cells bycolor-coded pHLA multimers complexed with in-silico predicted peptidessecured high throughput.

The discovery of epitopes in self-TAA was based on alloreactive T cells.The concept that cancer cells can be targeted by T cells recognizingself-peptides presented by foreign HLA, was first described by Staussand colleagues (15-17). Although these and other studies have shown thatcertain peptides can be specifically recognized when presented onallogeneic HLA (8, 9, 12, 18-23), it seemed possible that theyrepresented exceptions, since there is also evidence thatallo-restricted cells are more promiscuous with regard to peptiderecognition (9, 18, 24-28). However, when assessing reactivity to alarge number of epitopes at the single cell level, we did not findevidence indicating that peptide recognition on foreign HLA generallyhas a low specificity. First, T cells stimulated by CD20 or MPO did notreact with large panels of irrelevant pHLA multimers. More importantly,functional responses were absent or very low when stimulating the CTLlines with a large panel of HLA-A2pos CD20neg/MPOneg target cells,representing different histologies and presenting thousands of peptidesfrom endogenously expressed antigens (29) in addition to a wide range ofallogeneic HLA molecules. In contrast, these target cells elicitedefficient CTL degranulation when induced to express the relevantantigens or loaded with peptide. Finally, the CTLs responded stronglywhen incubated with leukemia cells and other target cells naturallyexpressing CD20 or MPO. Importantly, the results did not only reflectthe characteristics of a few selected clones, but were representative ofCTL lines reactive with 13 different peptide targets derived from twoproteins. Collectively, the results from this study therefore supportthe view that specific peptide recognition on foreign HLA is the rulerather than the exception.

The adoptive transfer of donor-derived T cells to a patient inallogeneic hematopoietic stem cell transplantation can induce remissionof leukemia by so-called graft-versus-leukemia effects (30). Arequirement to avoid potentially detrimental graft-versus-host disease,is that the donor and recipient are HLA-matched. However, recent studiesshow that clinically beneficial T cells can be generated from a singledonor for adoptive transfer to multiple patients that are only partiallyHLA-matched (31). Surprisingly, third-party EBV-specific T cells caneliminate EBV-associated post-transplantation lymphoma in spite oflikely immune recognition and rejection of foreign HLA by the patientimmune system (31). Interestingly, there is no evidence that suchmismatched CTLs reactive to EBV (32-35) or cytomegalovirus (36) causegraft-versus-host disease. These results, and the results presentedhere, bear promise that antigen-specific allogeneic T cells reactive toself-TAA can be generated from a single HLA-A2neg donor for transfer tomultiple HLA-A2pos patients. Alternatively, their receptors may begenetically transferred to redirect patient T cells to the tumor(37-39).

Elimination of normal and malignant B cells by anti-CD20 antibodytreatment is well tolerated by patients. This indicates that thecytotoxic effects of CD20 specific T cells, although mechanisticallydifferent from that of antibodies, would also be tolerated. In contrast,the removal of myeloid cells in leukemia by MPOpos/HLA-A2pos specific Tcells would result in significant toxicity unless accompanied by anallogeneic hematopoietic stem cell transplant from an HLA-A2neg donor.Such transplants, mismatched for one major HLA antigen, are accepted forhigh-risk patients in the clinic today, and would allow re-treatment ofa potential relapse with MPO specific T cells while sparing thetransplanted cells. The outlined platform for high-throughput epitopediscovery provides an important solution to the first bottleneck in theclinical translation of the described concept, and warrants furthertesting of safety and efficacy in vivo.

By use of a novel technology platform we have identified a 36 novelcytotoxic T cell epitopes from two self-tumor associated antigens(self-TAA). For a high number of these—and for all epitopes tested—wehave also demonstrated their potential utility as targets of cancerimmunotherapy directly.

Example 2

The method described in Example 1 above and illustrated in FIG. 4 wasused to test various peptide epitopes from Hepatitis C Virus (HCV)proteins. Monocyte-derived DCs (moDCs) from HLA-A2^(pos) donors weretransfected with mRNA encoding full-length HCV non-structural (NS)protein 3 or core Ag. The donors were HCV seronegative to ensure T cellrepertoires that were unbiased by previous infection. The moDCs wereco-cultured with autologous CD8^(pos) T cells. To assess theepitope-coverage of the reactive T cells, we prepared a panel ofcolor-coded peptide-HLA-A2 (pHLA) multimers containing 20 differentpeptides. Fifteen corresponded to previously characterized epitopes,whereas five were potentially novel epitopes predicted to be strongbinders to HLA-A*02:01 by the computer algorithm NetMHC (IC50<50 nM)(Table 3). The T cells were probed with mixtures of pHLA multimersfluorescing in different colors (FIG. 8 A,B).

Collectively, the CTLs that were generated from two donors covered themajority of the tested specificities. This included all of the nineknown epitopes in HCV core Ag, and seven out of six known and five novelepitopes in NS3 (FIG. 8 A,B). In addition to indicating a high degree ofefficiency in presenting a multitude of epitopes, the pattern ofmultimer labelling suggested specificity for the cognate targets. First,the large majority of T cells bound a single type of multimer only (FIG.8A). Second, T cells from HLA-B*08:01 or HLA-B*07:02 positive donorsselectively bound multimers of the respective HLA molecules. Third, Tcells stimulated by NS3 did not bind multimers containing peptides fromcore Ag, and vice versa (FIG. 8B).

Next, we examined the efficiency of the approach for detection of theHCV-derived epitopes when presented on foreign HLA. The describedexperiments were repeated using cells from HLA-A2^(neg) donors. ThemoDCs were transfected with HLA-A2, which we have demonstrated is anefficient strategy for peptide presentation on allogeneic HLA (10), andco-transfected with HCV antigens prior to co-culture with autologous Tcells. The results showed that HLA-A2^(neg) T cells largely recognizedthe same epitopes as T cells from HLA-A2^(pos) donors. Importantly,there was low cross-reactivity to irrelevant pHLA-A2 multimers (FIG.8C,D). Furthermore, freshly isolated T cells from the same donorsstained negatively with all multimers (data not shown). Thus, asrevealed by pHLA multimer staining, peptides bound to a foreign HLAmolecule appeared to be recognized with similar efficiencies andspecificities as those bound to self-HLA class I molecules.

TABLE 3Peptides predicted to bind HLA-A*02:01, -B*08:01 and -B*07:02 from HCV Core and NS3 antigensPredicted Peptide affinity # Position Sequence (nM) ReferenceCore-Hepatitis C viral antigen C1 177-185 FLLALLSCL     9Jackson, M., et al. J Med. Virol. 1999; 58: 239-46 (SEQ ID NO: 51) C277-85 AQPGYPWPL    25Anthony D. D., et al. Clin. Immunol. 2002; 103: 264-76 (SEQ ID NO: 52)C3 181-189 LLSCLTVPA    35Urbani S., et al. Hepatology. 2002; 33: 1533-43 (SEQ ID NO: 53) C4168-176 NLPGCSFSI    36 Löhr H. F., et al. J Hepatol. 1998; 29: 524-32(SEQ ID NO: 54) C5 35-44 YLLPRRGPRL   123Cerny A., et al. J Clin Invest. 1995; 95: 521-30 (SEQ ID NO: 55) C6132-140 DLMGYIPLV    14 Cerny A., et al. J Clin Invest. 1995; 95: 521-30(SEQ ID NO: 56) C7 178-187 LLALLSCLTV    24Cerny A., et al. J Clin Invest. 1995; 95: 521-30 (SEQ ID NO: 57) C841-49 GPRLGVRAT(B7)    34Koziel M. J., et al. J Virol. 1993; 67: 7522-32 (SEQ ID NO: 58) C9111-119 DPRRRSRNL(B7)    19Healey C., et al. Gastroenterology. 2002; 110: 1209 (SEQ ID NO: 59)NS3-Hepatitis C viral antigen NS3-1 44-52 FLATCINGV     6Wertheimer A. M., et al. Hepatology. 2003; 37: 577-89 (SEQ ID NO: 60)NS3-2 560-568 YLVAYQATV     7Wentworth P. A., et al. Int Immunology. 1996; 8: 651-659 (SEQ ID NO: 61)NS3-3 517-525 YMNTPGLPV    17 (SEQ ID NO: 62) NS3-4 581-589 QMWKCLIRL   17 (SEQ ID NO: 63) NS3-5 425-433 SVIDCNTCV    25 (SEQ ID NO: 64)NS3-6 14-22 LLGCIITSL    32 (SEQ ID NO: 65) NS3-7 439-447 FSLDPTFTI   42 (SEQ ID NO: 66) NS3-8 48-56 CINGVCWTV(SEQ    66Cerny A. et al. J Clin Invest. 1995; 95: 521-30 (SEQ ID NO: 67) NS3-9262-271 TGSPITYSTY 34458Rosen H. R. et al. Transplantation. 2002; 74: 209-16 (SEQ ID NO: 68)NS3-10 370-378 HSKKKCDEL(B8)  2240Koziel M. J. et al. J Clin Invest. 1995; 96: 2311-21 (SEQ ID NO: 69)NS3-11 586-594 LIRLKPTLH(B8) 15525Wong D. K. et al. J Virol. 2001; 75: 1229-35 (SEQ ID NO: 70)

REFERENCES

-   1. Andersen R S, et al. (2012) Dissection of T-cell antigen    specificity in human melanoma. Cancer Res 72(7):1642-1650.-   2. Admon A & Bassani-Sternberg M (2011) The Human Immunopeptidome    Project, a suggestion for yet another postgenome next big thing. Mol    Cell Proteomics 10(10):O111 011833.-   3. Rammensee H G, Falk K, & Rotzschke O (1993) Peptides naturally    presented by MHC class I molecules. Annual review of immunology    11:213-244.-   4. Stevanovic S & Schild H (1999) Quantitative aspects of T cell    activation—peptide generation and editing by MHC class I molecules.    Seminars in immunology 11(6):375-384.-   5. Hassan C, et al. (2013) The human leukocyte antigen-presented    ligandome of B lymphocytes. Mol Cell Proteomics.-   6. Nielsen M, et al. (2007) NetMHCpan, a method for quantitative    predictions of peptide binding to any HLA-A and -B locus protein of    known sequence. PloS one 2(8):e796.-   7. de Verteuil D, Granados D P, Thibault P, & Perreault C (2012)    Origin and plasticity of MHC I-associated self peptides.    Autoimmunity reviews 11(9):627-635.-   8. Stronen E, et al. (2009) Dendritic cells engineered to express    defined allo-HLA peptide complexes induce antigen-specific cytotoxic    T cells efficiently killing tumor cells. Scand J Immunol    69(4):319-328.-   9. Wilde S, et al. (2009) Dendritic cells pulsed with RNA encoding    allogeneic MHC and antigen induce T cells with superior antitumor    activity and higher TCR functional avidity. Blood 114(10):2131-2139.-   10. Shiina T, Hosomichi K, Inoko H, & Kulski J K (2009) The HLA    genomic loci map: expression, interaction, diversity and disease.    Journal of human genetics 54(1):15-39.-   11. Kessler J H & Melief C J (2007) Identification of T-cell    epitopes for cancer immunotherapy. Leukemia 21(9):1859-1874.-   12. Amir A L, et al. (2011) Allo-HLA-reactive T cells inducing    graft-versus-host disease are single peptide specific. Blood    118(26):6733-6742.-   13. Hadrup S R, et al. (2009) Parallel detection of antigen-specific    T-cell responses by multidimensional encoding of MHC multimers. Nat    Methods 6(7):520-526.-   14. Boczkowski D, Nair S K, Snyder D, & Gilboa E (1996) Dendritic    cells pulsed with RNA are potent antigen-presenting cells in vitro    and in vivo. J Exp Med 184(2):465-472.-   15. Sadovnikova E, Jopling L A, Soo K S, & Stauss H J (1998)    Generation of human tumor-reactive cytotoxic T cells against    peptides presented by non-self HLA class I molecules. Eur J Immunol    28(1):193-200.-   16. Savage P, et al. (2004) Use of B cell-bound HLA-A2 class I    monomers to generate high-avidity, allo-restricted CTLs against the    leukemia-associated protein Wilms tumor antigen. Blood    103(12):4613-4615.-   17. Gao L, Downs A M, & Stauss H J (2005) Immunotherapy with CTL    restricted by nonself MHC. Methods in molecular medicine    109:215-228.-   18. Felix N J, et al. (2007) Alloreactive T cells respond    specifically to multiple distinct peptide-MHC complexes. Nat Immunol    8(4):388-397.-   19. Housset D & Malissen B (2003) What do TCR-pMHC crystal    structures teach us about MHC restriction and alloreactivity? Trends    in immunology 24(8):429-437.-   20. Udaka K, Tsomides T J, & Eisen H N (1992) A naturally occurring    peptide recognized by alloreactive CD8+ cytotoxic T lymphocytes in    association with a class I MHC protein. Cell 69(6):989-998.-   21. Kronig H, et al. (2009) Allorestricted T lymphocytes with a high    avidity T-cell receptor towards NY-ESO-1 have potent anti-tumor    activity. Int J Cancer 125(3):649-655.-   22. Abrahamsen I W, et al. (2012) T cells raised against allogeneic    HLA-A2/CD20 kill primary follicular lymphoma and acute lymphoblastic    leukemia cells. Int J Cancer 130(8):1821-1832.-   23. Abrahamsen I W, et al. (2010) Targeting B cell leukemia with    highly specific allogeneic T cells with a public recognition motif.    Leukemia 24(11):1901-1909.-   24. Guimezanes A, et al. (2001) Identification of endogenous    peptides recognized by in vivo or in vitro generated alloreactive    cytotoxic T lymphocytes: distinct characteristics correlated with    CD8 dependence. Eur J Immunol 31(2):421-432.-   25. Mazza C, et al. (2007) How much can a T-cell antigen receptor    adapt to structurally distinct antigenic peptides? The EMBO journal    26(7):1972-1983.-   26. Leisegang M, et al. (2010) MHC-restricted fratricide of human    lymphocytes expressing survivin-specific transgenic T cell    receptors. The Journal of clinical investigation 120(11):3869-3877.-   27. Falkenburg W J, et al. (2011) Allogeneic HLA-A*02-restricted    WT1-specific T cells from mismatched donors are highly reactive but    show off-target promiscuity J Immunol 187(5):2824-2833.-   28. Stanislawski T, et al. (2001) Circumventing tolerance to a human    MDM2-derived tumor antigen by TCR gene transfer. Nat Immunol    2(10):962-970.-   29. Engelhard V H, Brickner A G, & Zarling A L (2002) Insights into    antigen processing gained by direct analysis of the naturally    processed class I MHC associated peptide repertoire. Molecular    immunology 39(3-4):127-137.-   30. Kolb H J (2008) Graft-versus-leukemia effects of transplantation    and donor lymphocytes. Blood 112(12):4371-4383.-   31. Cooper L J (2010) Off-the-shelf T-cell therapy. Blood    116(23):4741-4743.-   32. Barker J N, et al. (2010) Successful treatment of EBV-associated    posttransplantation lymphoma after cord blood transplantation using    third-party EBV-specific cytotoxic T lymphocytes. Blood    116(23):5045-5049.-   33. Wynn R F, et al. (2005) Treatment of    Epstein-Barr-virus-associated primary CNS B cell lymphoma with    allogeneic T-cell immunotherapy and stem-cell transplantation. The    lancet oncology 6(5):344-346.-   34. Hague T, et al. (2002) Treatment of Epstein-Barr-virus-positive    post-transplantation lymphoproliferative disease with partly    HLA-matched allogeneic cytotoxic T cells. Lancet 360(9331):436-442.-   35. Hague T, et al. (2007) Allogeneic cytotoxic T-cell therapy for    EBV-positive posttransplantation lymphoproliferative disease:    results of a phase 2 multicenter clinical trial. Blood    110(4):1123-1131.-   36. Melenhorst J J, et al. (2010) Allogeneic virus-specific T cells    with HLA alloreactivity do not produce GVHD in human subjects. Blood    116(22):4700-4702.-   37. Schumacher T N (2002) T-cell-receptor gene therapy. Nat Rev    Immunol 2(7):512-519.-   38. Morgan R A, et al. (2006) Cancer regression in patients after    transfer of genetically engineered lymphocytes. Science    314(5796):126-129.-   39. Robbins P F, et al. (2011) Tumor regression in patients with    metastatic synovial cell sarcoma and melanoma using genetically    engineered lymphocytes reactive with NY-ESO-1. Journal of clinical    oncology: official journal of the American Society of Clinical    Oncology 29(7):917-924.-   40. Lundegaard C, et al. (2008) NetMHC-3.0: accurate web accessible    predictions of human, mouse and monkey MHC class I affinities for    peptides of length 8-11. Nucleic acids research 36(Web Server    issue):W509-512.-   41. Toebes M, et al. (2006) Design and use of conditional MHC class    I ligands. Nat Med 12(2):246-251.-   42. Walchli S, et al. (2011) A practical approach to T-cell receptor    cloning and expression. PloS one 6(11):e27930.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

What is claimed is:
 1. A method of screening for novel T cell epitopesand T cell receptors binding to said novel epitopes, comprising: a)expressing a polypeptide comprising one or more candidate self-antigensderived from a self-protein in an antigen presenting cell that expressesa defined HLA molecule by introducing a nucleic acid construct encodingsaid polypeptide into said antigen presenting cell; b) utilizing anaffinity-based algorithmic prediction to identify a plurality ofpeptides from said polypeptide that are predicted to bind to saiddefined HLA molecule to provide predicted peptides; c) synthesizing saidpredicted peptides and contacting said defined HLA molecule with saidsynthesized predicted peptides to allow those peptides among saidsynthesized predicted peptides that exhibit binding to said defined HLAmolecule to generate predicted peptide:defined HLA molecule complexes;d) forming a plurality of labelled complexes of said synthesizedpredicted peptide:defined HLA molecules from those predicted peptidesthat bind to said defined HLA molecules to provide a plurality oflabelled predicted peptide:defined HLA molecule multimer complexes; e)obtaining donor T cells from a donor, wherein said defined HLA moleculeis not expressed by said T cells; f) contacting said antigen-presentingcells with said donor T cells to provide induced T cells; and g)identifying one or more of said plurality of said labelled predictedpeptide:defined HLA molecule multimer complexes binding to said donor Tcells to identify multimer complexes containing novel epitopes thatsimultaneously bind said defined HLA molecule and T-cell receptors onsaid induced T cells based on detection of said label.
 2. The method ofclaim 1, wherein said defined HLA molecule is naturally expressed insaid antigen presenting cell.
 3. The method of claim 1, wherein saiddefined HLA molecule is exogenously expressed in said antigen presentingcell.
 4. The method of claim 1, wherein said defined HLA molecule isHLA-A*02:01.
 5. The method of claim 1, wherein said nucleic acidconstruct is an mRNA construct.
 6. The method of claim 1, wherein saidnucleic acid construct is part of a viral vector.
 7. The method of claim1, wherein at least one of the one or more candidate self antigens isCD20 or myeloperoxidase.
 8. The method of claim 1, wherein the one ormore candidate self antigens is screened for cell-type specificexpression in normal and disease-affected cells prior to saidexpressing.
 9. The method of claim 1, wherein said forming comprisesUV-induced ligand exchange and multimerization.
 10. The method of claim1, wherein said donor T cells binding said predicted peptide:defined HLAmolecule complexes express a T-cell receptor that binds to said complexand wherein the method further comprises the step of cloning said T-cellreceptor from one of said identified T-cells and modifying cells from asubject to express said T-cell receptor to provide modified cells. 11.The method of claim 1, wherein said nucleic acid construct is a DNAconstruct.